attana - sensor technologies

Scientific Publications

  • mAbs, DOI: 10.1080/19420862.2017.1319023 Inhibition of HER3 activation and tumor growth with a human antibody binding to a conserved epitope formed by domain III and IVLisa C. Schmitt, Alexander Rau, Oliver Seifert, Jonas Honer, Meike Hutt, Simone Schmid, Jonas Zantow, Michael Hust, Stefan Dübel, Monilola A. Olayioye & Roland E. Kontermann

    Human epidermal growth factor receptor 3 (HER3, also known as ErbB3) has emerged as relevant target for antibody-mediated tumor therapy. Here, we describe a novel human antibody, IgG 3-43, recognizing a unique epitope formed by domain III and parts of domain IV of the extracellular region of HER3, conserved between HER3 and mouse ErbB3. An affinity of 11 nM was determined for the monovalent interaction. In the IgG format, the antibody bound recombinant bivalent HER3 with subnanomolar affinity (KD = 220 pM) and HER3-expressing tumor cells with EC50 values in the low picomolar range (27-83 pM). The antibody competed with binding of heregulin to HER3-expressing cells, efficiently inhibited phosphorylation of HER3 as well as downstream signaling, and induced receptor internalization and degradation. Furthermore, IgG 3-43 inhibited heregulin-dependent proliferation of several HER3-positive cancer cell lines and heregulin-independent colony formation of HER2-overexpressing tumor cell lines. Importantly, inhibition of tumor growth and prolonged survival was demonstrated in a FaDu xenograft tumor model in SCID mice. These findings demonstrate that by binding to the membrane-proximal domains III and IV involved in ligand binding and receptor dimerization, IgG 3-43 efficiently inhibits activation of HER3, thereby blocking tumor cell growth both in vitro and in vivo.

    © 2017 Taylor & Francis Group. All rights reserved.

  • Carbohydrate Polymers, Online 3 February 2017, In press Temperature effect on the complex formation between Pluronic F127 and starchYana Petkova-Olsson, , Samuel Altun, Henrik Ullsten and Lars Järnström

    In this study a systematic investigation of the temperature effect on the interactions between Pluronic F127 and hydroxypropylated oxidised potato starch by surface tension titrations and quartz crystal microbalance (QCM) analysis is presented. The binary mixture examined was subjected to 20 °C and 30 °C and the results indicated no presence of binary complexes at the lower temperature. However, at elevated temperature, an ability for inclusion complex formation was detected by the here used independent techniques. The formed inclusion complexes at 30 °C are presumably a product of hydrophobic interaction between Pluronic F127 and starch, where starch acts as a host molecule and Pluronic F127 due to its increased hydrophobicity is the guest molecule in this complex.

    © 2017 Elsevier Ltd. All rights reserved.

  • Nature: Sci. Rep. 7, 43006; doi: 10.1038/srep43006 (2017) Cellular glycosylation affects Herceptin binding and sensitivity of breast cancer cells to doxorubicin and growth factorsDiluka Peiris, Alexander F. Spector, Hannah Lomax-Browne, TayebehAzimi, Bala Ramesh, Marilena Loizidou, Hazel Welch and Miriam V. Dwek

    Alterations in protein glycosylation are a key feature of oncogenesis and have been shown to affect cancer cell behaviour perturbing cell adhesion, favouring cell migration and metastasis. This study investigated the effect of N-linked glycosylation on the binding of Herceptin to HER2 protein in breast cancer and on the sensitivity of cancer cells to the chemotherapeutic agent doxorubicin (DXR) and growth factors (EGF and IGF-1). The interaction between Herceptin and recombinant HER2 protein and cancer cell surfaces (on-rate/off-rate) was assessed using a quartz crystal microbalance biosensor revealing an increase in the accessibility of HER2 to Herceptin following deglycosylation of cell membrane proteins (deglycosylated cells Bmax: 6.83Hz; glycosylated cells Bmax: 7.35Hz). The sensitivity of cells to DXR and to growth factors was evaluated using an MTT assay. Maintenance of SKBR-3 cells in tunicamycin (an inhibitor of N-linked glycosylation) resulted in an increase in sensitivity to DXR (0.1μM DXR P<0.001) and a decrease in sensitivity to IGF-1 alone and to IGF-1 supplemented with EGF (P<0.001). This report illustrates the importance of N-linked glycosylation in modulating the response of cancer cells to chemotherapeutic and biological treatments and highlights the potential of glycosylation inhibitors as future combination treatments for breast cancer.

    © The Authors 2017 under license from Springer Nature

  • Chem. Commun., 20th September 2016, 52, 12326 Spatially well-defined carbohydrate nanoplatforms: synthesis, characterization and lectin interaction studyB. J. J. Timmer, M. Abellán Flos, L. Mønster Jørgensen, D. Proverbio, S. Altun, O. Ramström, T. Aastrup and S. P. Vincent

    Two novel dodecasubstituted carbohydrate nanoplatforms based on molecular Borromean rings and dodecaamine cages have been prepared for use in evaluating the importance of the spatial distribution of carbohydrates in their interaction with lectins. The binding affinities of the glyconanoplatforms were characterized using quartz crystal microbalance technology and compared with a monovalent reference and dodecaglycosylated fullerenes.

    © The Royal Society of Chemistry 2016

  • Analyst, 2016, 141, 3993-3996 Signal enhancement in ligand–receptor interactions using dynamic polymers at quartz crystal microbalance sensorsGunnar Dunér, Henrik Anderson, Zhichao Pei, Björn Ingemarsson, Teodor Aastrup and Olof Ramström

    The signal enhancement properties of QCM sensors based on dynamic, biotinylated poly(acrylic acid) brushes has been studied in interaction studies with an anti-biotin Fab fragment. The poly(acrylic acid) sensors showed a dramatic increase in signal response with more than ten times higher signal than the carboxyl-terminated self-assembled monolayer surface.

    © The Royal Society of Chemistry 2016

  • Analytical Chemistry, May 6, 2016 Combined Bacteria Microarray and Quartz Crystal Microbalance Approach for Exploring Glycosignatures of Nontypeable Haemophilus influenzae and Recognition by Host LectinsIoanna Kalograiaki, Begoña Euba, Davide Proverbio, María A. Campanero-Rhodes, Teodor Aastrup, Junkal Garmendia and Dolores Solís

    Recognition of bacterial surface epitopes by host receptors plays an important role in the infectious process and is intimately associated with bacterial virulence. Delineation of bacteria−host interactions commonly relies on the detection of binding events between purified bacteria- and host-target molecules. In this work, we describe a combined microarray and quartz crystal microbalance (QCM) approach for the analysis of carbohydrate-mediated interactions directly on the bacterial surface, thus preserving the native environment of the bacterial targets. Nontypeable Haemophilus influenzae (NTHi) was selected as a model pathogenic species not displaying a polysaccharide capsule or O-antigen-containing lipopolysaccharide, a trait commonly found in several important respiratory pathogens. Here, we demonstrate the usefulness of NTHi microarrays for exploring the presence of carbohydrate structures on the bacterial surface. Furthermore, the microarray approach is shown to be efficient for detecting strain-selective binding of three innate immune lectins, namely, surfactant protein D, human galectin-8, and Siglec-14, to different NTHi clinical isolates. In parallel, QCM bacteria-chips were developed for the analysis of lectin-binding kinetics and affinity. This novel QCM approach involves capture of NTHi on lectin-derivatized chips followed by formaldehyde fixation, rendering the bacteria an integrated part of the sensor chip, and subsequent binding assays with label-free lectins. The binding parameters obtained for selected NTHi-lectin pairs provide further insights into the interactions occurring at the bacterial surface.

    © 2016 American Chemical Society

  • Sensing and Bio-Sensing Research 9 (2016) 23–30 Real-time and label free determination of ligand binding-kinetics to primary cancer tissue specimens; a novel tool for the assessment of biomarker targetingThomas Mandel Clausen, Marina Ayres Pereira, Htoo Zarni Oo, Mafalda Resende, Tobias Gustavson, Yang Mao, Nobuo Sugiura, Janet Liew, Ladan Fazli, Thor G. Theander, Mads Daugaard, Ali Salanti

    In clinical oncology, diagnosis and evaluation of optimal treatment strategies are mostly based on histopathological examination combined with immunohistochemical (IHC) expression analysis of cancer-associated antigens in formalin fixed paraffin-embedded (FFPE) tissue biopsies. However, informative IHC analysis depends on both the specificity and affinity of the binding reagent, which are inherently difficult to quantify in situ. Here we describe a label-free method that allows for the direct and real-time assessment of molecular binding kinetics in situ on FFPE tissue specimens using quartz crystal microbalance (QCM) enabled biosensor technology. We analysed the interaction between the rVAR2 protein and its placental-like chondroitin sulfate (pl-CS) receptor in primary human placenta tissue and in breast and prostate tumour specimens in situ. rVAR2 interacted with FFPE human placenta and cancer tissue with an affinity in the nanomolar range, and showed no detectable interaction with pl-CS negative normal tissue. We further validated the method by including analysis with the androgen receptor N-20 antibody (anti-AR). As the KD value produced by this method is independent of the number of epitopes available, this readout offers a quantitative and unbiased readout for in situ binding-avidity and amount of binding epitopes. In summary, this method adds a new and important dimension to classical IHC-based molecular pathology by adding information about the binding characteristics in biologically relevant conditions. This can potentially be used to select optimal biologics for diagnostic and for therapeutic applications as well as guide the development of novel high affinity binding drugs.

    © 2016 The Authors. Published by Elsevier B.V.

  • Biosensors and Bioelectronics, Volume 76, 15 February 2016, Pages 113–130 Glyconanomaterials for biosensing applicationsNanjing Hao, Kitjanit Neranon, Olof Ramström and Mingdi Yan

    Nanomaterials constitute a class of structures that have unique physiochemical properties and are excellent scaffolds for presenting carbohydrates, important biomolecules that mediate a wide variety of important biological events. The fabrication of carbohydrate-presenting nanomaterials, glyconanomaterials, is of high interest and utility, combining the features of nanoscale objects with biomolecular recognition. The structures can also produce strong multivalent effects, where the nanomaterial scaffold greatly enhances the relatively weak affinities of single carbohydrate ligands to the corresponding receptors, and effectively amplifies the carbohydrate-mediated interactions. Glyconanomaterials are thus an appealing platform for biosensing applications. In this review, we discuss the chemistry for conjugation of carbohydrates to nanomaterials, summarize strategies, and tabulate examples of applying glyconanomaterials in in vitro and in vivo sensing applications of proteins, microbes, and cells. The limitations and future perspectives of these emerging glyconanomaterials sensing systems are furthermore discussed.

    Copyright © 2015 Elsevier B.V. All rights reserved.

  • Applied Surface Science, Volume 343, 15 July 2015, Pages 166–171 Ionization of covalent immobilized poly(4-vinylphenol) monolayers measured by ellipsometry, QCM and SPRSuji Uppalapati, Na Kong, Oscar Norberg, Olof Ramström and Mingdi Yan

    Covalently immobilized poly(4-vinylphenol) (PVP) monolayer films were fabricated by spin coating PVP on perfluorophenyl azide (PFPA)-functionalized surfaces followed by UV irradiation. The pH-responsive behavior of these PVP ultrathin films was evaluated by ellipsometry, quartz crystal microbalance (QCM) and surface plasmon resonance (SPR). By monitoring the responses of these films to pH in situ, the ionization constant of the monolayer thin films was obtained. The apparent pKa value of these covalently immobilized PVP monolayers, 13.4 by SPR, was 3 units higher than that of the free polymer in aqueous solution.

    Copyright © 2015 Elsevier B.V. All rights reserved.

  • Polymer Chemistry, DOI: 10.1039/c5py01954k Facile fabrication of glycopolymer-based iron oxide nanoparticles and their applications in the carbohydrate–lectin interaction and targeted cell imagingChen Shao, Xueming Li, Zhichao Pei, Dongdong Liu, Lin Wang, Hai Dong and
    Yuxin Pei

    A novel method for facile fabrication of glycopolymer-based iron oxide nanoparticles (GIONs) is developed. Via perfluorophenylazide photochemically induced C–H insertion, alkynyl groups were introduced onto the polymer which was precoated on the iron oxide nanoparticle surface. GIONs were then prepared by conjugating the azide-functionalized carbohydrate to the introduced alkynyl groups via click chemistry. Polyvinyl alcohol-coated and dextran-coated iron oxide NPs were chosen as scaffolds to attach two different carbohydrates, α-D-mannose and β-D-glucose, to fabricate multivalent GIONs, respectively. The multivalent GIONs demonstrated high binding affinities towards the corresponding lectins in both protein and cell chips. As a proof of concept, fluorescent GIONs (Gal-RhB-IONPs) were fabricated, which showed selective and efficient internalization by ASGP-R overexpressing HepG2 cells targeted.

    © The Royal Society of Chemistry 2016

  • Advanced Materials 2015 Sep 16;27(35):5165-70 Biomimetic Approach for Ion Channels Based on Surfactant Encapsulated Spherical Porous Metal-Oxide CapsulesMahon E., Garai S., Müller A. and Barboiu M.

    Distinguished hybrid clusters with hydrophilic and hydrophobic interiors embedded within cationic surfactant shells are spontaneously inserted into lipid bilayers, showing well-defined ionic conductance behaviors. The transport via the narrow pore gates acting as selectivity filters is controlled by the dehydration energy of the cations.

    © WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

  • mAbs. 2015 Oct 29:0. [Epub ahead of print] Pharmacokinetic properties of IgG and various Fc fusion proteins in mice.Unverdorben F, Richter F, Hutt M, Seifert O, Malinge P, Fischer N and Kontermann RE

    Fusion to an IgG Fc region is an established strategy to extend the half-life of therapeutic proteins. Most Fc fusion proteins, however, do not achieve the long half-life of IgGs. Based on findings that scFv-Fc fusion proteins exhibit a shorter half-life than the corresponding IgG molecules, we performed a comparative study of different antibody-derived Fc fusion proteins. We could confirm that fusion of single-chain Fv (scFv) and single-chain diabody (scDb) molecules to an Fc region yields in fusion proteins with substantially extended half-lives compared with the single-chain versions. However, even fusion proteins with a size similar to that of IgG, e.g., scDb-Fc, did not have a half-life as long as an IgG molecule. Binding to the neonatal Fc receptor (FcRn) under acidic and neutral conditions was similar for IgG and all Fc fusion proteins. However, we observed differences between IgG and the Fc fusion proteins for dissociation of FcRn-bound proteins induced by shifting from acidic to neutral pH, reflecting the physiological release mechanism, further supporting a contribution of the kinetics of pH-dependent release from FcRn to the pharmacokinetic properties of IgG and Fc fusion proteins.

  • Sensors and Actuators B: Chemical Volume 224, 1 March 2016, Pages 814–822 Oriented and reversible immobilization of His-tagged proteins on two- and three-dimensional surfaces for study of protein–protein interactions by a QCM biosensorXueming Li, Siyu Song, Yuxin Pei, Hai Dong, Teodor Aastrup and Zhichao Pei

    A two-dimensional (2D) and a three-dimensional (3D) His-tag capture surfaces were fabricated for oriented and reversible immobilization of His-tagged proteins on quartz crystal microbalance (QCM) biosensor surfaces, which can be used for label-free and real-time detection of the interactions between His-tagged protein and its interacting protein (analyte). His-tagged proteins immobilized on the 2D His-tag capture surface maintained a higher binding activity than those immobilized on a 2D carboxyl surface via amine coupling. The 3D His-tag capture surface has about twice the amount of immobilization capacity as the 2D His-tag capture surface, which enables a higher sensitivity for detection. His-tag capture surface can be optionally regenerated to remove the His-tagged protein as well as the analyte for the next cycle of His-tagged protein immobilization, or to only selectively remove the analyte, leaving the His-tagged protein on the surface for the next cycle of analyte binding. Furthermore, the kinetic and affinity studies of the interactions between the His-tagged protein and its interacting protein were performed. This study provides an efficient way to study protein–protein interactions by oriented and reversible immobilization of His-tagged proteins on QCM biosensor surfaces.

    Copyright © 2017 Elsevier B.V. or its licensors or contributors.

  • Cancer Cell, Volume 28, Issue 4, p500–514, 12 October 2015 Targeting Human Cancer by a Glycosaminoglycan Binding Malaria ProteinAli Salanti, T M Clausen, M Ø. Agerbæk, N Al Nakouzi, M Dahlbäck, H Z Oo, S Lee, T Gustavsson, J R. Rich, B J. Hedberg, Y Mao, L Barington, M A. Pereira, J LoBello, M Endo, L Fazli, J Soden, C K. Wang, A F. Sander, R Dagil, S Thrane, P J. Holst, L Meng, F Favero, G J. Weiss, M A. Nielsen, J Freeth, T O. Nielsen, J Zaia, N L. Tran, J Trent, J S. Babcook, T G. Theander, P H. Sorensen, M Daugaard

    Plasmodium falciparum engineer infected erythrocytes to present the malarial protein, VAR2CSA, which binds a distinct type chondroitin sulfate (CS) exclusively expressed in the placenta. Here, we show that the same CS modification is present on a high proportion of malignant cells and that it can be specifically targeted by recombinant VAR2CSA (rVAR2). In tumors, placental-like CS chains are linked to a limited repertoire of cancer-associated proteoglycans including CD44 and CSPG4. The rVAR2 protein localizes to tumors in vivo and rVAR2 fused to diphtheria toxin or conjugated to hemiasterlin compounds strongly inhibits in vivo tumor cell growth and metastasis. Our data demonstrate how an evolutionarily refined parasite-derived protein can be exploited to target a common, but complex, malignancy-associated glycosaminoglycan modification.

    © 2015 Elsevier Inc.

  • PLOS ONE, DOI:10.1371, journal.pone.0139838, October 2, 2015 A Fab-Selective Immunoglobulin-Binding Domain from Streptococcal Protein G with Improved Half-Life Extension PropertiesFelix Unverdorben, Meike Hutt, Oliver Seifert and Roland E. Kontermann

    Half-life extension strategies have gained increasing interest to improve the pharmacokinetic and pharmacodynamic properties of protein therapeutics. Recently, we established an immunoglobulin-binding domain (IgBD) from streptococcal protein G (SpGC3) as module for half-life extension. SpGC3 is capable of binding to the Fc region as well as the CH1 domain of Fab arms under neutral and acidic conditions. Using site-directed mutagenesis, we generated a Fab-selective mutant (SpGC3Fab) to avoid possible interference with the FcRn-mediated recycling process and improved its affinity for mouse and human IgG by site-directed mutagenesis and phage display selections. In mice, this affinity-improved mutant (SpGC3FabRR) conferred prolonged plasma half-lives compared with SpGC3Fab when fused to small recombinant antibody fragments, such as singlechain Fv (scFv) and bispecific single-chain diabody (scDb). Hence, the SpGC3FabRR

    domain seems to be a suitable fusion partner for the half-life extension of small recombinant therapeutics. The half-life extension properties of SpGC3 can be retained by restricting binding to the Fab fragment of serum immunoglobulins and can be improved by increasing binding activity. The modified SpGC3 module should be suitable to extend the half-life of therapeutic proteins and, thus to improve therapeutic activity.

    ©2015 Unverdorben et al

  • Nanoscale, 2015,7, 16039-16045 Weighing the surface charge of an ionic liquidNicklas Hjalmarsson, Daniel Wallinder, Sergei Glavatskih, Rob Atkin, Teodor Aastrup and Mark W. Rutland

    Electrochemical quartz crystal microbalance has been used to measure changes in the composition of the capacitive electrical double layer for 1-ethyl-3-methylimidazolium tris(pentafluoroethyl)-trifluorophosphate, an ionic liquid, in contact with a gold electrode surface as a function of potential. The mass difference between the cation and anion means that the technique can effectively “weigh” the surface charge accurately with high temporal resolution. This reveals quantitatively how changing the potential alters the ratio of cations and anions associated with the electrode surface, and thus the charge per unit area, as well as the kinetics associated with these interfacial processes. The measurements reveal that it is diffusion of co-ions into the interfacial region rather than expulsion of counterions that controls the relaxation. The measured potential dependent double layer capacitance experimentally validates recent theoretical predictions for counterion overscreening (low potentials) and crowding (high potentials) at electrode surfaces. This new capacity to quantitatively measure ion composition is critical for ionic liquid applications ranging from batteries, capacitors and electrodeposition through to boundary layer structure in tribology, and more broadly provides new insight into interfacial processes in concentrated electrolyte solutions.

    © Royal Society of Chemistry 2017

  • Scientific Reports 5, Article number: 14066 (2015) Real-time and label-free analysis of binding thermodynamics of carbohydrate-protein interactions on unfixed cancer cell surfaces using a QCM biosensorXueming Li, Siyu Song, Qi Shuai, Yihan Pei, Teodor Aastrup, Yuxin Pei & Zhichao Pei

    A novel approach to the study of binding thermodynamics and kinetics of carbohydrate-protein interactions on unfixed cancer cell surfaces using a quartz crystal microbalance (QCM) biosensor was developed, in which binding events take place at the cell surface, more closely mimicking a biologically relevant environment. In this study, colon adenocarcinoma cells (KM-12) and ovary adenocarcinoma cells (SKOV-3) grew on the optimized polystyrene-coated biosensor chip without fixation. The association and dissociation between the cell surface carbohydrates and a range of lectins, including WGA, Con A, UEA-I, GS-II, PNA and SBA, were monitored in real time and without label for evaluation of cell surface glycosylation. Furthermore, the thermodynamic and kinetic parameters of the interaction between lectins and cell surface glycan were studied, providing detailed information about the interactions, such as the association rate constant, dissociation rate constant, affinity constant, as well as the changes of entropy, enthalpy and Gibbs free energy. This application provides an insight into the cell surface glycosylation and the complex molecular recognition on the intact cell surface, which may have impacts on disease diagnosis and drug discovery.

  • Tetrahedron; Volume 71, Issue 23, 10 June 2015, Pages 4023–4030 Synthesis and binding affinity analysis of positional thiol analogs of mannopyranose for the elucidation of sulfur in different positionBin Wu, Jiantao Ge, Bo Ren, Zhichao Pei and Hai Dong

    Synthetic routes towards thio-α/β-d-mannose derivatives are presented. Double parallel or double serial inversion was successfully applied in the efficient synthesis of 2-thio- or 2,4-di-thio-mannoside derivatives. The protein recognition properties of the synthesized positional thiol analogs of mannose were then evaluated in a competition binding assay with the model lectin Concanavalin A (Con A), in order to investigate the roles of thiol group in the different position of the mannopyranose ring in binding affinity. Though the substitution of oxygen atom with sulfur atom in the methyl α-d-mannoside ring usually displayed low or no binding affinity towards Con A, it was a surprise finding that the methyl 2-thio-α-d-mannoside displayed four times higher inhibition than methyl α-d-mannoside, indicating the particular importance of 2-position for modification of α-d-mannoside. Methyl 3-thio-α-d-mannoside also displayed inhibition towards Con A, indicating that the O-atom at the C-3 position is less important in the binding site.

    Copyright © 2015 Elsevier Ltd. All rights reserved.

  • Scientist Live; 8th June 2015 Investigating manufactured nanoparticlesTeodor Aastrup, Diluka Peiris and Daniel Wallinder

    Manufactured nanoparticles (MNPs) are increasingly being considered for use in biomedical applications ranging from drug delivery to cellular imaging. Thus, the understanding of MNPs’ interactions with biological systems has become vital for both their safety profile and efficient applications. The growing interest on elucidating the impact of physicochemical properties of NPs (eg, size, surface charge, hydrophobicity, or shape) on their subsequent cellular interactions necessitates the exploration of new technical tools.

    © The author(s). Courtesy of Setform Limited.

  • Sensors 2015, 15, 5884-5894; doi:10.3390/s150305884 Study of the Interaction of Trastuzumab and SKOV3 Epithelial Cancer Cells Using a Quartz Crystal Microbalance SensorLouise Elmlund, Camilla Käck, Teodor Aastrup and Ian A. Nicholls

    Analytical methods founded upon whole cell-based assays are of importance in early stage drug development and in fundamental studies of biomolecular recognition. Here we have studied the binding of the monoclonal antibody trastuzumab to human epidermal growth factor receptor 2 (HER2) on human ovary adenocarcinoma epithelial cancer cells (SKOV3) using quartz crystal microbalance (QCM) technology. An optimized procedure for immobilizing the cells on the chip surface was established with respect to fixation procedure and seeding density. Trastuzumab binding to the cell decorated sensor surface was studied, revealing a mean dissociation constant, KD, value of 7 ± 1 nM (standard error of the mean). This study provides a new perspective on the affinity of the antibody-receptor complex presented a more natural context compared to purified receptors. These results demonstrate the potential for using whole cell-based QCM assay in drug development, the screening of HER2 selective antibody-based drug candidates, and for the study of biomolecular recognition. This real time, label free approach for studying interactions with target receptors present in their natural environment afforded sensitive and detailed kinetic information about the binding of the analyte to the target.

    ©2015 MDPI AG (Basel, Switzerland)

  • mAbs, Volume 7, 2015 - Issue 1 Preclinical and early clinical development of GNbAC1, a humanized IgG4 monoclonal antibody targeting endogenous retroviral MSRV-Env proteinFrançois Curtin, Hervé Perron, Arno Kromminga, Hervé Porchet and Alois B Lang

    Monoclonal antibodies (mAbs) play an increasing important role in the therapeutic armamentarium against multiple sclerosis (MS), an inflammatory and degenerative disorder of the central nervous system. Most of the mAbs currently developed for MS are immunomodulators blocking the inflammatory immune process. In contrast with mAbs targeting immune function, GNbAC1, a humanized IgG4 mAb, targets the multiple sclerosis associated retrovirus envelope (MSRV-Env) protein, an upstream factor in the pathophysiology of MS. MSRV-Env protein is of endogenous retroviral origin, expressed in MS brain lesions, and it is pro-inflammatory and toxic to the remyelination process, by preventing the differentiation of oligodendrocyte precursor cells. We present the preclinical and early clinical development results of GNbAC1. The specificity of GNbAC1 for its endogenous retroviral target is described. Efficacy of different mAb versions of GNbAC1 were assessed in MSRV-Env induced experimental allergic encephalitis (EAE), an animal model of MS. Because the target MSRV-Env is not expressed in animals, no relevant animal model exists for a proper in vivo toxicological program. An off-target 2-week toxicity study in mice was thus performed, and it showed an absence of safety risk. Additional in vitro analyses showed an absence of complement or antibody-dependent cytotoxicity as well as a low level of cross-reactivity to human tissues. The first-in-man clinical study in 33 healthy subjects and a long-term clinical study in 10 MS patients showed that GNbAC1 is well tolerated in humans without induction of immunogenicity and that it induces a pharmacodynamic response on MSRV biomarkers. These initial results suggest that the mAb GNbAC1 could be a safe long-term treatment for patients with MS with a unique therapeutic mechanism of action.

    © 2017 The Author(s). Published with license by Taylor & Francis Group, LLC

  • J. Agric. Food Chem. 2013, 61, 11400−11409 Inducement of Cytokine Release by GFPBW2, a Novel Polysaccharide from Fruit Bodies of Grifola frondosa, through Dectin‐1 in MacrophagesYing Wang, Jianping Fang, Xinyan Ni, Jie Li, Qin Liu, Qun Dong, Jinyou Duan, and Kan Ding

    Polysaccharides, especially β-glucans isolated from various species of mushrooms, are considered as biological response modifiers (BRMs) to be widely used in the treatment of cancer, especially due to their immunostimulatory activity. We herein characterized the structure of a novel water-soluble homogeneous polysaccharide (GFPBW2) from the fruit bodies of mushroom Grifola frondosa and investigated its immunomodulatory activity in vitro. GFPBW2 was purified from the alkali-extracted fractions by stepwise elution with a molecular weight of 26.2 kDa. On the basis of infrared and NMR spectroscopy, methylation and monosaccharide composition analysis, partial acid hydrolysis, and Smith degradation, its structure was elucidated to possess a backbone consisting of β-D-1,3- and β-D-1,4-linked glucopyranosyl residues, with branches attached to O-6 of β-D-1,3-linked glucopyranosyl residues. Functionally, it is an effective inducer of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) secretion in murine resident peritoneal macrophages. Using quartz crystal microbalance (QCM) analysis, we found that GFPBW2 could bind dendritic cell-associated C-type lectin-1 (Dectin-1) with an affinity constant (Kd) value of 1.08 × 10 −7 M, while it could activate Syk and enhance TNF-α production in RAW264.7 cells overexpressing wild type but not mutant Dectin-1. Furthermore, Syk, NF-κB signaling, and cytokine release in resident peritoneal macrophages induced by GFPBW2 could be significantly inhibited by a specific Dectin-1 blocking reagent, Laminarin. These data suggested that GFPBW2 might be a potential ligand of Dectin-1, and the potential of GFPBW2 to activate macrophage through triggering cytokine secretion might be attributed, at least in part, to the involvement of Dectin-1.

    © 2013 American Chemical Society

  • Molecular Pharmacology Fast Forward, July 7, 2015 as DOI: 10.1124/mol.115.099671 Ligand residence time at GPCRs – why we should take our time to study itC. Hoffmann, M. Castro, A. Rinken, R. Leurs, S.J. Hill, H.F. Vischer

    Over the past decade the kinetics of ligand binding to a receptor have received increasing interest. The concept of drug-target residence time is becoming an invaluable parameter for drug optimization. It holds great promise for drug-development and its optimization is thought to reduce off-target effects. The success of long-acting drugs like tiotropium support this hypothesis. Nonetheless, we know surprisingly little about the dynamics and the molecular detail of the drug binding process. Since protein dynamics and adaptation during the binding event will change the conformation of the protein, ligand binding will not be the static process that is often described. This can cause problems since simple mathematical models often fail to adequately describe the dynamics of the binding process. In this perspective we will discuss the current situation with an emphasis on GPCRs. This are important membrane protein drug-targets that undergo conformational changes upon agonist binding in order to communicate signalling information across the plasma membrane of cells.

    Copyright ©2015 by the American Society for Pharmacology and Experimental Therapeutics

  • International Pharmaceutical Industry, 2014 Vol. 6, Issue 2, 54-57 Label-free Cell-based Assay for the Characterization of Peptide Receptor InteractionsWright, Proverbio, Valnohova, Schulte and Aastrup

    Both the drug development process and fundamental studies of mechanisms’ underlying biological interactions require analytical methods that approximate the in vivo situation as much as possible. This can be achieved by using label-free cell-based assays, since they eliminate non-natural treatment, e.g. purification and labelling, of both the target receptor and the drug candidate molecule.

    In this paper, we present a new label-free assay that we have developed for characterising interactions between peptides and their target receptors in a cellular environment. We will describe one such interaction that was assessed by monitoring the binding profile in real-time and by quantifying its kinetic rate constants for association and dissociation, as well as its affinity. In addition, the absence of off-target interactions with the cell membrane was confirmed by analysis of the binding profile.

     IPI © 2014 International Pharmaceutical Industry

  • Nucleic Acids Research, 2014-10-01 doi: 10.1093/nar/gku882 Highly potent dUTPase inhibition by a bacterial repressor protein reveals a novel mechanism for gene expression controlJudit E. Szabó, Veronika Németh, Veronika Papp-Kádár, Kinga Nyíri, Ibolya Leveles, Ábris Á. Bendes, Imre Zagyva, Gergely Róna, Hajnalka L. Pálinkás, Balázs Besztercei, Olivér Ozohanics, Károly Vékey, Károly Liliom, Judit Tóth and Béata G. Vértessy

    Transfer of phage-related pathogenicity islands of Staphylococcus aureus (SaPI-s) was recently reported to be activated by helper phage dUTPases.

    This is a novel function for dUTPases otherwise involved in preservation of genomic integrity by sanitizing the dNTP pool. Here we investigated the molecular mechanism of the dUTPase-induced gene expression control using direct techniques. The expression of SaPI transfer initiating proteins is repressed by proteins called Stl. We found that 11 helper phage dUTPase eliminates SaPIbov1 Stl binding to its cognate DNA by binding tightly to Stl protein. We also show that dUTPase enzymatic activity is strongly inhibited in the dUTPase:Stl complex and that the dUTPase:dUTP complex is inaccessible to the Stl repressor. Our results disprove the previously proposed G-protein-like mechanism of SaPI transfer activation. We propose that the transfer only occurs if dUTP is cleared from the nucleotide pool, a condition promoting genomic stability of the virulence elements.


    © The Authors 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  • THE JOURNAL OF NUCLEAR MEDICINE 2014; 55:452–459 Quantitative ImmunoPET of Prostate Cancer Xenografts with 89Zr- and 124I-Labeled Anti-PSCA A11 MinibodyScott M. Knowles, Kirstin A. Zettlitz, Richard Tavaré, Matthew M. Rochefort, Felix B. Salazar, David B. Stout, Paul J. Yazaki, Robert E. Reiter, and Anna M. Wu

    Prostate stem cell antigen (PSCA) is expressed on the cell surface in 83%–100% of local prostate cancers and 87%–100% of prostate cancer bone metastases. In this study, we sought to develop immunoPET agents using 124I- and 89Zr-labeled anti-PSCA A11 minibodies (scFv-CH3 dimer, 80 kDa) and evaluate their use for quantitative immunoPET imaging of prostate cancer. Methods: A11 anti-PSCA minibody was alternatively labeled with 124I- or 89Zr-desferrioxamine and injected into mice bearing either matched 22Rv1 and 22Rv1· PSCA or LAPC-9 xenografts. Small-animal PET data were obtained and quantitated with and without recovery coefficient–based partial volume correction, and the results were compared with ex vivo biodistribution. Results: Rapid and specific localization to PSCA-positive tumors and high-contrast imaging were observed with both 124I and 89Zr-labeled A11 anti-PSCA minibody. However, the differences in tumor uptake and background uptake of the radiotracers resulted in different levels of imaging contrast. The nonresidualizing 124I-labeled minibody had lower tumor uptake (3.62 ± 1.18 percentage injected dose per gram [%ID/g] 22Rv1·PSCA, 3.63 ± 0.59 %ID/g LAPC-9) than the residualizing 89Zr-labeled minibody (7.87 ± 0.52 %ID/g22Rv1·PSCA, 9.3360.87%ID/gLAPC-9,P<0.0001 for each), but the 124I-labeled minibody achieved higher imaging contrast because of lower nonspecific uptake and better tumor–to–soft-tissue ratios (22Rv1·PSCA:22Rv1 positive-to-negative tumor, 13.31 ± 5.59 124I-A11 and 4.87 ± 0.52 89Zr-A11, P=0.02). Partial-volume correction was found to greatly improve the correspondence between small-animal PET and ex vivo quantification of tumor uptake for immunoPET imaging with both radionuclides. Conclusion: Both 124I and 89Zr-labeled A11 anti-PSCA minibody showed high-contrast imaging of PSCA expression in vivo. However, the 124I-labeled A11 minibody was found to be the superior imaging agent because of lower nonspecific uptake and higher tumor–to–soft-tissue contrast. Partial volume correctionwas found to be essential for robust quantification of immunoPET imaging with both 124I- and 89Zr-labeled A11 minibody.

  • ACS Applied Materials & Interfaces: One-Step Synthesis of Dual Clickable Nanospheres via Ultrasonic- Assisted Click Polymerization for Biological ApplicationsYong Hou, Shoupeng Cao, Xueming Li, Beibei Wang, Yuxin Pei, Lin Wang, and Zhichao Pei

    Dual clickable nanospheres (DCNSs) were synthesized in one step using an efficient approach of ultrasonic-assisted azide−alkyne click polymerization, avoiding the need of surfactants. This novel approach presents a direct clickable monomer-to-nanosphere synthesis. Field emission scanning electron microscopy (FESEM), Fourier transform infrared spectroscopy (FTIR), and dynamic laser scattering (DLS) were used to characterize the synthesized DCNSs. Numerous terminal alkynyl and azide groups on the surface of DCNSs facilitate effective conjugation of multiple molecules or ligands onto a single nanocarrier platform under mild conditions. To exemplify the potential of DCNSs in biological applications, (1) multivalent glyco-nanoparticles (GNPs) were prepared by clicking DCNSs with azide-functionalized and alkyne-functionalized lactose sequentially for the determination of carbohydrate-galectin interactions with quartz crystal microbalance (QCM) biosensor. Using protein chip (purified galectin-3 coated on chip) and cell chip (Jurkat cells immobilized on chip), the QCM sensorgrams showed excellent binding activity of GNPs for galectins; (2) fluorescent GNPs were prepared by clicking DCNSs with azide-functionalized Rhodamine B and alkyne functionalized lactose sequentially in order to target galectin, which is over expressed on the surface of Jurkat cells. The fluorescent images obtained clearly showed the cellular internalization of fluorescent GNPs. This fluorescent probe could be easily adapted to drugs to construct lectin-targeted drug delivery systems. Thus, DCNSs prepared with our method may provide a wide range of potential applications in glycobiology and biomedicine.

    © 2014 American Chemical Society

  • mAbs 6:2, 533–546; March/April 2014 3D modeling and characterization of the human CD115 monoclonal antibody H27K15 epitope and design of a chimeric CD115 targetBenoît Grellier, Fabrice Le Pogam, Marc Vitorino, Jean-Philippe Starck, Michel Geist, Vanessa Duong, Hélène Haegel, Thierry Menguy, Jean-Yves Bonnefoy, Jean-Baptiste Marchand, and Philippe Ancian

    The humanized monoclonal antibody H27K15 specifically targets human CD115, a type III tyrosine kinase receptor involved in multiple cancers and inflammatory diseases. Binding of H27K15 to hCD115 expressing cells inhibits the functional effect of colony-stimulating factor-1 (CSF-1), in a non-competitive manner. Both homology modeling and docking programs were used here to model the human CD115 extracellular domains, the H27K15 variable region and their interaction. The resulting predicted H27K15 epitope includes mainly the D1 domain in the N-terminal extracellular region of CD115 and some residues of the D2 domain. Sequence alignment with the non-binding murine CD115, enzyme-linked immunosorbent assay, nuclear magnetic resonance spectroscopy and affinity measurements by quartz crystal microbalance revealed critical residues of this epitope that are essential for H27K15 binding. A combination of computational simulations and biochemical experiments led to the design of a chimeric CD115 carrying the human epitope of H27K15 in a murine CD115 backbone that is able to bind both H27K15 as well as the murine ligands CSF-1 and IL-34. These results provide new possibilities to minutely study the functional effects of H27K15 in a transgenic mouse that would express this chimeric molecule.

    © 2014 Landes Bioscience

  • Anal Bioanal Chem 4 April 2014, DOI 10.1007/s00216-014-7821-9 Partial-filling affinity capillary electrophoresis and quartz crystal microbalance with adsorption energy distribution calculations in the study of biomolecular interactions with apolipoprotein E as interaction partnerLipponen, Tähkä, Samuelsson,Jauhiainen, Metso, Cilpa-Karhu, Fornstedt, Kostiainen, Riekkola

    Adsorption energy distribution (AED) calculations were successfully applied to partial-filling affinity capillary electrophoresis (PF-ACE) to facilitate more detailed studies of biomolecular interactions. PF-ACE with AED calculations was employed to study the interactions between two isoforms of apolipoprotein E (apoE) and dermatan sulfate (DS), and a quartz crystal microbalance (QCM) was used in combination with AED calculations to examine the interactions of the 15-amino-acid peptide fragment of apoE with DS. The heterogeneity of the interactions was elucidated. Microscale thermophoresis was used to validate the results. The interactions studied are of interest because, in vivo, apolipoprotein E localizes on DS-containing regions in the extracellular matrix of human vascular subendothelium. Two-site binding was demonstrated for the isoform apoE3 and DS, but only one-site binding for apoE2–DS. Comparable affinity constants were obtained for the apoE2–DS, apoE3–D3, and 15-amino-acid peptide of apoE–DS using the three techniques. The results show that combining AED calculations with modern biosensing techniques can open up another dimension in studies on the heterogeneity and affinity constants of biological molecules.

    Copyright © Springer-Verlag Berlin Heidelberg 2014

  • Biosensors 2014, 4(2), 137-149 A Phage Display Screening Derived Peptide with Affinity for the Adeninyl MoietyElmlund, Söderberg, Suriyanarayanan, Nicholls

    Phage display screening of a surface-immobilized adenine derivative led to the identification of a heptameric peptide with selectivity for adenine as demonstrated through quartz crystal microbalance (QCM) studies. The peptide demonstrated a concentration dependent affinity for an adeninyl moiety decorated surface (KD of 968 ± 53.3 μM), which highlights the power of piezoelectric sensing in the study of weak interactions.

    © 2014 Elmlund et al.

  • Chem. Commun., 2014, 50, 731-733 Entropy-driven lectin-recognition of multivalent glycovesiclesZineb Mouline, Eugene Mahon, Emeline Gomez, Veronique Barragan-Montero, Jean-Louis Montero and Mihail Barboiu

    Multivalent glycovesicle recognition over lectin layers emphasizes effects on the dynamic lateral fluidity of glycoside clusters upon multivalent binding at the bilayer surface and vice versa.

    Chem. Commun., 2014, 50, 731

    © The Royal Society of Chemistry 2013

  • Analytical Biochemistry 443 (2013) 139–147 Three complementary techniques for the clarification of temperature effect on low-density lipoprotein–chondroitin-6-sulfate interactionCilpa-Karhu, Lipponen, Samuelsson, Öörni, Fornstedt, Riekkola

    A rigorous processing of adsorption data from quartz crystal microbalance technology was successfully combined with the data obtained by partial filling affinity capillary electrophoresis and molecular dynamics for the clarification of the temperature effect on the interaction of a major glycosaminoglycan chain chondroitin-6-sulfate (C6S) of proteoglycans with low-density lipoprotein (LDL) and with a peptide fragment of apolipoprotein B-100 (residues 3359–3377 of LDL, PPBS). Two experimental techniques and computational atomistic methods demonstrated a nonlinear pattern of the affinity of C6S at temperatures above 38.0 C to both LDL and PPBS. The temperature affects the interaction of C6S with LDL and PPBS by influencing the structural behavior of glycosaminoglycan C6S and/or that of LDL.

    © 2013 Elsevier Inc. All rights reserved.

  • Biosens Bioelectron. 2014 Mar 15;53:154-9 Atmospheric pressure plasma polymers fortuned QCM detection of protein adhesionRusu, Asandulesa, Topala, Pohoata, Dumitrascu, Barboiu

    Our efforts have been concentrated in preparing plasma polymeric thin layers at atmospheric pressure grown on Quartz Crystal Microbalance-QCM electrodes for which the non-specific absorption of proteins can be efficiently modulated, tuned and used for QCM biosensing and quantification. Plasma polymerization reaction at atmospheric pressure has been used as a simple and viable method for the preparation of QCM bioactive surfaces, featuring variable protein binding properties. Polyethyleneglycol (ppEG), polystyrene (ppST) and poly(ethyleneglycol-styrene) (ppST-EG) thin-layers have been grown on QCM electrodes. These layers were characterized by Atomic Force Microscopy (AFM), Contact angle measurements, Fourier transform infrared (FTIR) and X-ray photoelectron spectroscopy (XPS). The plasma ppST QCM electrodes present a higher adsorption of Concanavalin A (ConA) and Bovine Serum Albumin (BSA) proteins when compared with the commercial coated polystyrene (ppST) ones. The minimum adsorption was found for ppEG,

    surface, known by their protein anti-fouling properties. The amount of adsorbed proteins can be tuned by the introduction of PEG precursors in the plasma discharge during the preparation of ppST polymers.

    © 2013 Elsevier B.V. All rights reserved.

  • J Nanobiotechnology. 2014 Mar 21;12:8 Biotin selective polymer nano-filmsElmlund, Suriyanarayanan, Wiklander, Aastrup, Nicholls

    BACKGROUND: The interaction between biotin and avidin is utilized in a wide range of assay and diagnostic systems. A robust material capable of binding biotin should offer scope in the development of reusable assay materials and biosensor recognition elements.

    RESULTS: Biotin-selective thin (3-5 nm) films have been fabricated on hexadecanethiol self assembled monolayer (SAM) coated Au/quartz resonators. The films were prepared based upon a molecular imprinting strategy where N,N'-methylenebisacrylamide and 2-acrylamido-2-methylpropanesulfonic acid were copolymerized and grafted to the SAM-coated surface in the presence of biotin methyl ester using photoinitiation with physisorbed benzophenone. The biotinyl moiety selectivity of the resonators efficiently differentiated biotinylated peptidic or carbohydrate structures from their native counterparts.

    CONCLUSIONS: Molecularly imprinted ultra thin films can be used for the selective recognition of biotinylated structures in a quartz crystal microbalance sensing platform. These films are stable for periods of at least a month. This strategy should prove of interest for use in other sensing and assay systems.

    © 2014 Elmlund et al.; licensee BioMed Central Ltd.

  • Chem. Eur. J. 2014, 20, 1–7 Multivalent Recognition of Concanavalin A by {Mo132} Glyconanocapsules-Toward Biomimetic Hybrid MultilayersBarboiu, Mouline, Silion, Licsandru, Simionescu, Mahon, Pinteala

    Herein, we consider M_ller’s spherical, porous, anionic, molybdenum oxide based capsule, (NH4)42- [{(MoVI)MoVI

    5O21(H2O)6}12{MoV2O4(CH3COO)}30]•10CH3COONH4•300H2O_(NH4)42•1a•crystal ingredients_1, {Mo132}, as an effective sugar-decorated nanoplatform for multivalent lectin recognition. The ion-exchange of NH4+ ions of 1 with cationic-

    sugars, d-mannose-ammonium chloride (2) or d-glucoseammonium chloride (3) results in the formation of glyconanocapsules (NH4)42_n2n•1a and (NH4)42_m3m•1a. The Mannose (NH4)42_n2n•1a capsules bind selectively Concanavalin A (Con A) in aqueous solution, giving an association avidity constant of Kmulti a =4.6_104m_1 and an enhancement factor of b=Kmulti a /Kmono ass =21.9, reminiscent of the formation of “glycoside clusters” on the external surface of glyconanocapsule. The glyconanocapsules (NH4)42_n2n•1a and (NH4)42_m3m•1a self-assemble in “hybrid multilayers” by successive layer-bylayer deposition of (NH4)42_n2n•1a or (NH4)42_m3m•1a and Con A. These architectures, reminiscent of versatile mimics of artificial tissues, can be easily prepared and quantified by using quartz crystal microgravimetry (QCM). The “biomimetic hybrid multilayers” described here are stable under a continual water flow and they may serve as artificial networks for a greater depth of understanding of various biological mechanisms, which can directly benefit the fields of chemical separations, sensors or storage-delivery devices.

    © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  • mAbs 5:5, 736–747; September/October 2013 A unique anti-CD115 monoclonal antibody which inhibits osteolysis and skews human monocyte differentiation from M2-polarized macrophages toward dendritic cellsHélène Haegel, Christine Thioudellet, Rémy Hallet, Michel Geist, Thierry Menguy, Fabrice Le Pogam, Jean-Baptiste Marchand, Myew-Ling Toh, Vanessa Duong, Alexandre Calcei, Nathalie Settelen, Xavier Preville, Marie Hennequi, Benoit Grellier, Philippe Ancian, Jukka Rissanen, Pascal Clayette, Christine Guillen, Ronald Rooke and Jean-Yves Bonnefoy

    Cancer progression has been associated with the presence of tumor-associated M2-macrophages (M2-TAMs) able to inhibit anti-tumor immune responses. It is also often associated with metastasis-induced bone destruction mediated by osteoclasts. Both cell types are controlled by the CD115 (CSF-1R)/colony-stimulating factor-1 (CSF-1, M-CSF) pathway, making CD115 a promising target for cancer therapy. Anti-human CD115 monoclonal antibodies (mAbs) that inhibit the receptor function have been generated in a number of laboratories. These mAbs compete with CSF-1 binding to CD115, dramatically affecting monocyte survival and preventing osteoclast and macrophage differentiation, but they also block CD115/CSF-1 internalization and degradation, which could lead to potent rebound CSF-1 effects in patients after mAb treatment has ended. We thus generated and selected a non-ligand competitive anti-CD115 mAb that exerts only partial inhibitory effects on CD115 signaling without blocking the internalization or the degradation of the CD115/CSF-1 complex. This mAb, H27K15, affects monocyte survival only minimally, but downregulates osteoclast differentiation and activity. Importantly, it inhibits monocyte differentiation to CD163+CD64+ M2-polarized suppressor macrophages, skewing their

    differentiation toward CD14−CD1a+ dendritic cells (DCs). In line with this observation, H27K15 also drastically inhibits monocyte chemotactic protein-1 secretion and reduces interleukin-6 production; these two molecules are known to be involved in M2-macrophage recruitment. Thus, the non-depleting mAb H27K15 is a promising anti-tumor candidate, able to inhibit osteoclast differentiation, likely decreasing metastasis-induced osteolysis, and able to prevent M2 polarization of TAMs while inducing DCs, hence contributing to the creation of more efficient anti-tumor immune responses.

    ©2013 Landes Bioscience

  • J. Biol. Chem. 2013, 288:995-1008 The C-terminal Peptide of Chondroadherin Modulates Cellular Activity by Selectively Binding to Heparan Sulfate ChainsLisbet Haglund, Viveka Tillgren, Patrik Önnerfjord and Dick Heinegård

    Chondroadherin, a leucine-rich repeat family member, contains a very C-terminal sequence CKFPTKRSKKAGRH359, now shown to bind to heparin with a KD of 13 µM. This observation led us to investigate whether chondroadherin interacts via this C-terminal heparin-binding domain with glycosaminoglycan chains of proteoglycans at the cell surface. Cells were shown to bind this heparin-binding peptide in FACS analysis, and the interaction was shown to be with glycosaminoglycans because it was abolished when sulfation was inhibited by chlorate treatment of the cells. In separate experiments, heparin and heparin sulfate inhibited the peptide interaction in a dose-dependent manner. Using a human chondrosarcoma and a murine osteoblast cell line, heparan sulfate proteoglycans were identified as the cell surface receptors involved in the binding. Different binding syndecans were identified in the two different cell lines, indicating that the same protein core of a proteoglycan may have structural and functional differences in the attached heparan sulfate chains. Upon binding to coated peptide, cells spread, demonstrating engagement of the cytoskeleton, but no focal adhesion complex was formed. The number of cells adhering via their β1 integrin receptor to collagen type II or chondroadherin was profoundly and rapidly enhanced by the addition of the heparin-binding peptide. The peptide added to the cells caused ERK phosphorylation, showing that it triggered intracellular signaling. The results show that heparan sulfate chains differ between various members of the proteoglycan families on a given cell, but also differ between the same proteoglycan on different cells with a potential for differential regulation of cellular activities.

    © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

  • Innovations in Pharmaceutical Technology, 2013, Issue 46, 48-51 Talking Sense – A combined biochemical and cell-based biosensorT. Aastrup

    The combined biochemical and cell-based biosensor enables detailed interaction characterization between biopharmaceutical molecules, such as antibodies, and target receptors on cell surfaces. This results in cell-based information on affinity and kinetic rate constants considering accessibility and clustering of receptors for the interactions, as well as revealing off-target interaction between the antibody and cell membrane and therapeutic accessibility of the receptors. 

    IPT online © 2013 The Pharmaceutical Technology Journal

  • PLoS ONE 8(8): e72156 August 19, 2013 Antagonistic TNF Receptor One-Specific Antibody (ATROSAB): Receptor Binding and In Vitro BioactivityRichter, Liebig, Guenzi, Herrmann, Scheurich, Pfizenmaier, Kontermann

    The epitope of ATROSAB resides in the N-terminal region of TNFR1 covering parts of CRD1 and CRD2. By site-directed mutagenesis, we identified Arg68 and His69 of TNFR1 as important residues for ATROSAB binding. ATROSAB inhibited binding of 125I-labeled TNF to HT1080 in the subnanomolar range. Furthermore, ATROSAB inhibited release of IL-6 and IL-8 from HeLa and HT1080 cells, respectively, induced by TNF or lymphotoxin alpha (LTα). Different from an agonistic antibody (Htr-9), which binds to a region close to the ATROSAB epitope but elicits strong TNFR1 activation, ATROSAB showed a negligible induction of IL-6 and IL-8 production over a broad concentration range. We further verified that ATROSAB, comprising mutations within the Fc region known to abrogate complement fixation and antibody-mediated cellular effector functions, indeed lacks binding activity for C1q, FcγRI (CD64), FcγRIIB (CD32b), and FcγRIII (CD16) disabling ADCC and CDC.

    © 2013 Richter et al.

  • Protein Engineering, Design & Selection, 8 August, 2013 An anti-TNFR1 scFv-HSA fusion protein as selective antagonist of TNF actionVerena Berger, Fabian Richter, Kirstin Zettlitz, Felix Unverdorben, Peter Scheurich, Andreas Herrmann, Klaus Pfizenmaier and Roland E. Kontermann

    IZI-06.1 is a humanized anti-TNFR1 single-chain fragment variable (scFv) that selectively inhibits binding of tumor necrosis factor (TNF) and lymphotoxin alpha to tumor necrosis factor receptor 1 (TNFR1) but not TNFR2. Recently, IZI-06.1 was converted into a fully human IgG1 antibody (ATROSAB) for the treatment of inflammatory diseases. Here, we compare the bivalent ATROSAB with a monovalent scFv-human serum albumin (HSA) fusion protein lacking any antibody-associated effector functions and possessing approximately only half the molecular mass of an IgG, which should facilitate accumulation in inflamed tissues. Furthermore, the half-life of the scFv should be strongly extended while maintaining monovalent binding, avoiding a possible signal transduction by receptor cross-linking in the absence of TNF. The scFv-HSA fusion protein was produced by stably transfected Chinese hamster ovary cells and purified by affinity chromatography. The fusion protein bound specifically to TNFR1 in enzyme-linked immunosorbent assay and TNFR1-transfected mouse embryonic fibroblasts. Affinity determined by quartz crystal microbalance was reduced compared with ATROSAB, which resulted also in a reduced inhibitory activity. Compared with the scFv fragment, the halflife of the fusion protein was significantly increased, although not reaching the long half-life of ATROSAB. In summary, the scFv-HSA may provide an alternative to the full-length IgG1 with the ability to selectively inhibit TNFR1 and exploiting the pharmacokinetic properties of albumin.

    Protein Engineering, Design & Selection vol. 26 no. 10 pp. 581–587, 2013

    © The Author 2013. Published by Oxford University Press

  • J Virol. 2013 Jul;87(13):7747-53 Protection by immunoglobulin dual-affinity retargeting antibodies against dengue virusBrien, Sukupolvi-Petty, Williams, Lam, Schmid, Johnson, Harris, Diamond

    Dengue viruses are the most common arthropod-transmitted viral infection, with an estimated 390 million human infections annually and ∼3.6 billion people at risk. Currently, there are no approved vaccines or therapeutics available to control the global dengue virus disease burden. In this study, we demonstrate the binding, neutralizing activity, and therapeutic capacity of a novel bispecific dual-affinity retargeting molecule (DART) that limits infection of all four serotypes of dengue virus.

    Copyright © 2013, American Society for Microbiology. All Rights Reserved.

  • Antibody Engineering and Therapeutics Conference - The Annual Meeting of the Antibody Society, Dec. 8-12, 2013, Huntington Beach, CA, USA Cell-Antibody Interaction Study Using Herceptin™ (trastuzumab) Binding to SKOV-3 Epithelial Cancer Cells Characterized by Attana™ Quartz Crystal Microbalance TechnologyL. Elmlund, C. Käck, L. Kaiser, T. Aastrup, I.A. Nicholls

    Overexpressed HER-2 (human epidermal growth factor 2) receptor is a problem in many cancer tumors. In fact, 20-30% of breast cancer tumors are caused by HER-2 receptor overexpression and the receptor is therefore used both as a biomarker and for treatment. Because of its poor prognosis early diagnosis of these tumors is of importance.

  • Journal of Colloid and Interface Science (2013) Protein-resistant hyperbranched polyethyleneimine brush surfacesSubramanian Suriyanarayanan, Hung-Hsun Lee, Bo Liedberg, Teodor Aastrup, Ian A. Nicholls.

    A novel hyperbranched polyethyleneimine (PEI) modified gold surface has been designed, fabricated, and investigated with respect to its ability to resist non-specific adsorption of proteins. The facile synthesis strategy, based on self-assembly, involves immobilization of polyethyleneimine to gold surfaces modified with 11-mercaptoundecanoic acid (MuDA) monolayers using traditional carbodiimide chemistry. The hyperbranched polymer brushes were characterized by X-ray photoelectron spectroscopy (XPS). Reflection absorption infrared spectroscopy (RAIRS) and ellipsometry measurements showed the thickness of
    the PEI brushes increases with adsorption solution ionic strength. Polymer brush surface concentrations can be improved from 2560 to 3880 chains/lm2 by changing the ionic strength of the adsorption solution (PBS) by varying NaCl concentration from 0 to 650 mM. Protein adsorption (pH 7.4) was evaluated under flow injection analysis (FIA) conditions using a quartz crystal microbalance (QCM). The PEI brushes suppress protein adsorption, for example, cytochrome C, bovine serum albumin (BSA), and ribonuclease A, to less than 0.08 lg/cm2 and the protein resistance increases with increasing ionic strength of the carrier solution, performance comparable to that achieved with comparable PEG-coated surfaces. The PEI brushes were exceptionally stable, with adsorption characteristics maintained after 6 months storage in aqueous conditions (pH 7.4, 25 C, PBS). The potential of hyperbranched PEI structures as protein-resistant surfaces is discussed.

  • ChemComm, 30 July 2013 A suspension-cell biosensor for real-time determination of binding kinetics of protein–carbohydrate interactions on cancer cell surfacesXueming Li, Yuxin Pei, Ruina Zhang, Qi Shuai, Feng Wang, Teodor Aastrup and Zhichao Pei

    A novel lectin-based suspension-cell biosensor for label-free determination of binding kinetics of protein–carbohydrate interactions on cancer cell surfaces using QCM is described. This cell-biosensor facilitates evaluation of glycosylation in real time on suspension cancer cell surfaces, where binding events take place, more closely mimicking a native environment compared with traditional biosensors.

    © The Royal Society of Chemistry 2013


  • Biosensors and Bioelectronics, 34 (2012) 51–56 Photogenerated lectin sensors produced by thiol-ene/yne photo-click chemistry in aqueous solutionOscar Norberg, Irene H. Lee, Teodor Aastrup, Mingdi Yan and Olof Ramström

    The photoinitiated radical reactions between thiols and alkenes/alkynes (thiol-ene and thiol-yne chemistry) have been applied to a functionalization methodology to produce carbohydrate-presenting surfaces for analyses of biomolecular interactions. Polymer-coated quartz surfaces were functionalized with alkenes or alkynes in a straightforward photochemical procedure utilizing perfluorophenylazide (PFPA) chemistry. The alkene/alkyne surfaces were subsequently allowed to react with carbohydrate thiols in water under UV-irradiation. The reaction can be carried out in a drop of water directly on the surface without photoinitiator, and any disulfide side products were easily washed away after the functionalization process. The resulting carbohydrate-presenting surfaces were evaluated in real-time studies of protein–carbohydrate interactions using a quartz crystal microbalance (QCM) flow-through system with recurring injections of selected lectins, with intermediate regeneration steps using low pH buffer. The resulting methodology proved fast, efficient and scalable to high-throughput analysis formats, and the produced surfaces showed significant protein binding with expected selectivities of the lectins used in the study.

    © 2012 Elsevier B.V.

  • Biosensors and Bioelectronics (2012)(In Press) A novel approach to determining the affinity of protein–carbohydrate interactions employing adherent cancer cells grown on a biosensor surfaceDiluka Peiris, Anatoliy Markiv, G. Paul Curley, Miriam V. Dwek

    The development of biological agents for the treatment of solid tumours is an area of considerable activity. We are pursuing carbohydrate-binding proteins (lectins) in a strategy aimed at targeting cancer associated changes in glycosylation. To evaluate lectin–cancer cell interactions we developed a novel cell biosensor in which binding events take place at the cell surface, more closely mimicking an in vivo system. Metastatic, SW620, and non-metastatic, SW480, colorectal cancer cells were grown on the surface of a tissue-culture compatible polystyrene coated biosensor chip and housed in a quartz crystal microbalance (QCM) apparatus, the kinetics of binding of a diverse range of lectins was evaluated. The lectin Helix pomatia agglutinin (HPA) has been shown to bind aggressive metastatic cancer and was produced
    in recombinant form (His- and RFP-tagged). The affinity of HPA was in the nanomolar range to the metastatic SW620 cells but was only in the micromolar range to the non-metastatic SW480. Overall, the dissociation constant (KD) of the lectins tested in the new cell biosensor system was an order of magnitude lower (nanomolar range) than has generally been reported with systems such as QCM/SPR. This new cell-biosensor enables molecular interactions to be studied in a more relevant environment. An intrinsic problem with developing new biological therapies is the difficulty in determining the affinity with which proteins will interact with intact cell surfaces. This methodology will be of interest to researchers developing new biological approaches for targeting cell surfaces in a wide range of diseases, including cancer.

    Crown Copyright © 2012 Published by Elsevier B.V.

  • Poster presented at Asian Communication for Glycobiology and Glycotechnology, Oct 2012 Cell-Based Biosensors: A Quartz Crystal Microbalance Approach to the Study of Carbohydrate-LectinInteractionsZhichaoPei, JulienSaint-Guirons, Camilla Käck, Bjorn Ingemarsson2, TeodorAastrup
  • ICS conference Sep 2012, Poland Searching by Affinity Ranking for the IC2 Monoclonal Autoantibody Targeted Autoantigenic Epitope on the Surface of Pancreatic Beta-CellsIda Dalgaard Pedersen, Kathrine Louise Jensen, Camilla Käck, Theodor Aastrup, Mickael Blaise, Anna Åkesson.


    Type 1 diabetes mellitus is a chronic disorder resulting from autoimmune destruction of insulin-producing pancreatic β-cells. In advance of clinical symptoms, autoantibodies are produced and self-reactive lymphocytes infiltrate the pancreas to destroy β-cells.


    Affinity measurements using QCM revealed a very high cellular affinity. Furthermore, binding of different formats of IC2 were measured to intact INS-1E cells, sonicated plasma membrane vesicles, and lipids, using the Q-Sense E4 quartz crystal
    microbalance with dissipation.

  • Doctoral thesis - KTH Royal Institute of Technology; Trita-CHE-Report, ISSN 1654-1081; TRITA-CHE Report 2012:49 Grafted Molecular Layers for Control of Surface PropertiesDunér, Gunnar

    The goal of this thesis work was to develop responsive surface grafted brushlayers for control of surface properties and to gain insights in the molecular mechanisms that control these properties. Three types of grafted layers were investigated, as outlined below. In the first system studied, poly(AAc) was synthesized by a grafting from approach, utilizing a photopolymerization reaction from a macroinitiator cast onto QCM substrates. The responsiveness in terms of frequency change, Δf, of the resulting brushes to changes in bulk pH was studied with QCM. Further, the friction properties of poly(AAc) was elucidated with colloidal probe AFM as a function of pH and counterion valency. High friction (μ=0.27) was found in presence of CaCl2 at high pH (7.5), but not under any other condition explored. It was concluded that the high friction was due to intralayer COO--Ca2+--OOC bridges. QCM-D was utilized for studying viscoelastic properties of growing poly(AAc) films during in situ photopolymerization. By Voigt modeling, the thickness, shear elasticity and shear viscosity were extracted. These parameters were observed to undergo sudden transitions at a critical thickness, and from this thickness the grafting density of the growing poly(AAc) layers was determined. In addition, the sensitivity to changes in Δf and ΔD with respect to the thickness of the poly(AAc) films was investigated, and the results showed that high sensitivity in ΔD is retained at higher film thicknesses than for Δf, and that the sensitivity with respect to noise can significantly alter the thickness that is best suited for the study of viscoelastic changes in sensor applications.

    © KTH Royal Institute of Technology 2012

  • Innovations in Pharmaceutical Technology - Issue 40(2012) (Online) A New Generation of Cell-BiosensorsMiriam Dwek and Teodor Aastrup

    A new cell-biosensor enables molecular interactions to be studied in a more relevant cellular environment and, as a model for understanding drug-cell interactions, provides a new biological approach for targeting cell surfaces in a wide range of diseases, including cancer.

    The full article is available free to view online:

    IPTonline © 2012 The Pharmaceutical Technology Journal

  • Drug Discovery and Development (05/14/2012) Quartz Crystal Microbalance Technology Provides Biologically Relevant DataTeodor Aastrup and Ingo Montenbruck

    The pharmaceutical and drug discovery communities are under ever greater pressure from the rising costs of R&D, increasing regulatory hurdles, the relatively low rate of annual drug approvals, and the number of blockbuster drugs going off-patent in the next few years. As a result, many companies are refocusing their drug discovery programs to provide a broader perspective of modes of action―as well as inclusion of biomarkers and wider use of early in vivo animal model pharmacology―offering a more predictive view of which candidates might have the greatest chance of success in the preclinical process and subsequent clinical trials.

    © 2012 Advantage Business Media

  • A biosensor-based concept for antibody-GPCR interaction studies using immobilized lipoparticlesSamuel Altun, Magnus Brändén, Teodor Aastrup, Torbjörn Pettersson

    This study demonstrates the applicability of the Attana A200 biosensor in combination with memLAYER immobilization for functional characterization of membrane proteins, such as GPCRs. Using the lipoparticle system the often time-consuming and expensive process of i) purifyingand ii) reconstituting the membrane protein of interest into liposomes for functional studies of membrane proteins are circumvented. The results also illustrate the benefits of immobilizing multiplelayers of lipoparticles using memLAYER, which increases the sensor surface density of the receptor and the response in subsequent ligand-receptor binding events.

  • International Journal of Nanomedicine, 27 Feb. 2012, 2012:7 905–914 Functionalization of single-walled carbon nanotubes and their binding to cancer cellsS.Y. Madani, A. Tan, M. Dwek, A. Seifalian

    Single-walled carbon nanotubes (SWCNTs) have novel properties including their nanoscale size and ease of cellular uptake. This makes them useful for drug delivery, and their photo-thermal effects make them potentially useful in a wide range of applications, particularly the treatment of solid tumors. The poor solubility of SWCNTs has, however, been an issue that may potentially limit the utility of SWCNTs for cancer treatment. Functionalization of the surface of the tubes may be an approach to overcome this problem.

    © Madani et al, publisher and licensee Dove Medical Press Ltd. 2012

  • Biosensors and Bioelectronics, 20 Feb. 2012 (Online) Real-time analysis of the carbohydrates on cell surfaces using a QCM biosensor: a lectin-based approachZ- Pei, J. Saint-Guirons, C. Käck, B. Ingemarsson, T. Aastrup

    A novel approach to the study of molecular interactions on the surface of mammalian cells using a QCM biosensor was developed. For this study, an epidermoid carcinoma cell line (A-431) and a breast adenocarcinoma cell line (MDA-MB-468) were immobilized onto polystyrene-coated quartz crystals. The binding and dissociation between the lectin Con A and the cells as well as the inhibition of the binding by monosaccharides were monitored in real time and provided an insight into the complex avidic recognition of cell glycoconjugates. The real-time lectin screening of a range of lectins, including Con A, DBA, PNA and UEA-I, enabled the accurate study of the glycosylation changes between cells, such as changes associated with cancer progression and development. Furthermore, the kinetic parameters of the interaction of Con A with MDA-MB-468 cells were studied. This application provides investigators in the field of glycobiology with a novel tool to study cell surface glycosylation and may also have impacts on drug discovery.

    © Elsevier B.V. 2012

  • Analytical Biochemistry 421 (2012) 351–361 Domain-based assays of individual antibody concentrations in an oligoclonal combination targeting a single proteinQ. Meng, M. Li, M.A. Silberg, F. Conrad, J. Bettencourt, R. To, C. Huang, J. Ma, K. Meyer, R. Shimizu, L. Cao, M.T. Tomic, J.D. Marks

    Quantitation of individual monoclonal antibodies (mAbs) within a combined antibody drug product is required for preclinical and clinical drug development, including pharmacokinetic (PK), toxicology, stability, and biochemical characterization studies of such drugs. We have developed an antitoxin, XOMA 3AB, consisting of three recombinant mAbs that potently neutralize the known subtypes of type A botulinum neurotoxin (BoNT/A). The three mAbs bind nonoverlapping BoNT/A epitopes with high affinity. XOMA 3AB is being developed as a treatment for botulism resulting from BoNT/A. To develop antibody-specific assays, we cloned, expressed, and purified BoNT/A domains from Escherichia coli. Each mAb bound only to its specific domain with affinity comparable to the binding to holotoxin. mAb-specific domains were used to develop an enzyme-linked immunosorbent assay (ELISA) for characterization of the integrity and binding activity of the three mAbs in the drug product. An electrochemiluminescence bridging assay that is robust to interference from components in serum was also developed, and we demonstrate that it can be used for PK assays. This type of antigen engineering to generate mAb-specific domains is a general method allowing quantitation and characterization of individual mAbs in a mAb cocktail that binds the same protein and is superior to anti-idiotype approaches.

    © 2011 Elsevier Inc.

  • Protein Eng Des Sel. 2012 Feb;25(2):81-8. Half-life extension of a single-chain diabody by fusion to domain B of staphylococcal protein AUnverdorben, Färber-Schwarz, Richter, Hutt, Kontermann

    Binding of a therapeutic protein to a long-circulating plasma protein can result in a strongly extended half-life. Among these plasma proteins, albumin and immunoglobulins are of special interest because of their exceptionally long half-life, which is to a great extent determined by recycling through the neonatal Fc receptor (FcRn). Many strategies have been established employing reversible binding to albumin, e.g. using an albumin-binding domain from streptococcal protein G. We show here that the half-life of a recombinant antibody molecule can also be prolonged by fusion to a single immunoglobulin-binding domain (IgBD) from staphylococcal protein A. This domain (domain B, SpA(B)) is composed of 56 amino acid residues and was fused to the C-terminus of a bispecific single-chain diabody (scDb). The scDb-SpA(B) fusion protein was produced in HEK293 cells and retained its antigen-binding activity as shown by enzyme-linked immunosorbent assay and flow cytometry. Furthermore, the fusion protein was capable of binding to human and mouse IgG in a pH-dependent manner. In mice, the terminal half-life of the fusion protein was improved from ∼1-2 h of the unmodified scDb to 11.8 h. Although the fusion protein did not reach the long half-life seen for IgG, our results established the applicability of a single bacterial IgBD for half-life extension purposes.

    © Unverdorben et al. 2012. Published by Oxford University Press. All rights reserved.

  • Vaccine, 2012 Jan 11;30(3):572-9 The effect of adjuvants on the immune response induced by a DBL4ɛ-ID4 VAR2CSA based Plasmodium falciparum vaccine against placental malariaPinto VV, Salanti A, Joergensen LM, Dahlbäck M, Resende M, Ditlev SB, Agger EM, Arnot DE, Theander TG, Nielsen MA.

    A vaccine protecting women against placental malaria could be based on the sub-domains of the VAR2CSA antigen, since antibodies against the DBL4ɛ-ID4 subunit of the VAR2CSA protein can inhibit parasite binding to the placental ligand chondroitin sulphate A (CSA). Here we tested the ability ofDBL4ɛ-ID4 to induce binding-inhibitory antibodies when formulated with adjuvants approved for human use. We have characterized the immune response of DBL4ɛ-ID4 in combination with Freund's complete and incomplete adjuvant and with three adjuvants currently being used in clinical trials: Montanide(®) ISA 720, Alhydrogel(®) and CAF01. Antibodies induced against DBL4ɛ-ID4 in combination with these adjuvants inhibited parasite binding to CSA from 82% to 99%. Although, different epitope recognition patterns were obtained for the different formulations, all adjuvant combinations induced strong Th1 and Th2 type responses, resulting in IgG with similar binding strength, with to the DBL4ɛ-ID4 antigen. These results demonstrate that the DBL4ɛ-ID4 antigen is highly immunogenic and that binding inhibitory antibodies are induced when formulated with any of the tested adjuvants.

    Copyright © 2011 Elsevier Ltd. All rights reserved.

  • Journal of Biological Chemistry, doi: 10.1074/jbc.M111.311522, December 6, 2011 Plasma half-life extension of small recombinant antibodies by fusion to immunoglobulin-binding domains (IgBD)Meike Hutt, Aline Färber-Schwarz, Felix Unverdorben, Fabian Richter & Roland E. Kontermann

    Many therapeutic proteins possessing a small size are rapidly cleared from circulation. Half-life extension strategies have therefore become increasingly important to improve the pharmacokinetic and pharmacodynamic properties of protein therapeutics. Here, we performed a comparative analysis of the half-life extension properties of various bacterial immunoglobulin-binding domains (IgBD) derived from Staphylococcus protein A (SpA), Streptococcus protein G (SpG) and Finegoldia (formerly Peptostreptococcus) protein L (PpL). These domains, composed of 50-60 amino acid residues, were fused to the C-terminus of a single-chain Fv (scFv) and a bispecific singlechain diabody (scDb), respectively. All fusion proteins were produced in mammalian cells and retained their antigen-binding properties. The half-lives of the antibody molecules were prolonged to varying extent for the different IgBDs. The strongest effects in mice were observed for domain C3 of SpG (SpGC3), followed by domains B and D of SpA, suggesting that SpGC3 is particularly useful to extend the plasma half-life of small proteins.

    © 2011 The American Society for Biochemistry and Molecular Biology, Inc.

  • PLoS ONE 6(9): e24558 2011, September 13, 2011 In Vitro Evolution of Allergy Vaccine Candidates, with Maintained Structure, but Reduced B Cell and T Cell Activation CapacityOla B. Nilsson, Justus Adedoyin, Claudio Rhyner, Theresa Neimert-Andersson, Jeanette Grundström, Kurt D. Berndt, Reto Crameri, Hans Grönlund

    Allergy and asthma to cat (Felis domesticus) affects about 10% of the population in affluent countries. Immediate allergic symptoms are primarily mediated via IgE antibodies binding to B cell epitopes, whereas late phase inflammatory reactions are mediated via activated T cell recognition of allergen-specific T cell epitopes. Allergen-specific immunotherapy relieves symptoms and is the only treatment inducing a long-lasting protection by induction of protective immune responses. The aim of this study was to produce an allergy vaccine designed with the combined features of attenuated T cell activation, reduced anaphylactic properties, retained molecular integrity and induction of efficient IgE blocking IgG antibodies for safer and efficacious treatment of patients with allergy and asthma to cat. The template gene coding for rFel d 1 was used to introduce random mutations, which was subsequently expressed in large phage libraries. Despite accumulated mutations by up to 7 rounds of iterative error-prone PCR and biopanning, surface topology and structure was essentially maintained using IgE-antibodies from cat allergic patients for phage enrichment. Four candidates were isolated, displaying similar or lower IgE binding, reduced anaphylactic activity as measured by their capacity to induce basophil degranulation and, importantly, a significantly lower T cell reactivity in lymphoproliferative assays compared to the original rFel d 1. In addition, all mutants showed ability to induce blocking antibodies in immunized mice.The approach presented here provides a straightforward procedure to generate a novel type of allergy vaccines for safer and efficacious treatment of allergic patients.

    © 2011 Nilsson et al.

  • J Biol Chem. 2011 Oct 14;286(41):35699-707 Unbinding of hyaluronan accelerates the enzymatic activity of bee hyaluronidaseIliás, Liliom, Greiderer-Kleinlercher, Reitinger, Lepperdinger

    Hyaluronan (HA), a polymeric glycosaminoglycan ubiquitously present in higher animals, is hydrolyzed by hyaluronidases (HAases). Here, we used bee HAase as a model enzyme to study the HA-HAase interaction. Located in close proximity to the active center, a bulky surface loop, which appears to obstruct one end of the substrate binding groove, was found to be functionally involved in HA turnover. To better understand kinetic changes in substrate interaction, binding of high molecular weight HA to catalytically inactive HAase was monitored by means of quartz crystal microbalance technology. Replacement of the delimiting loop by a tetrapeptide interconnection increased the affinity for HA up to 100-fold, with a K(D) below 1 nm being the highest affinity among HA-binding proteins surveyed so far. The experimental data of HA-HAase interaction were further validated showing best fit to the theoretically proposed sequential two-site model. Besides the one, which had been shown previously in course of x-ray structure determination, a previously unrecognized binding site works in conjunction with an unbinding loop that facilitates liberation of hydrolyzed HA.

    © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

  • J Immunol 2011; 186:959-968 Neonatal FcR Overexpression Boosts Humoral Immune Response in Transgenic MiceCervenak, Bender, Schneider, Magna, Valer Carstea, Liliom, Erdei, Bosze and Kacskovics

    The neonatal FcR (FcRn) regulates IgG and albumin homeostasis, mediates maternal IgG transport, takes active part in phagocytosis, and delivers Ag for presentation.We have previously shown that overexpression of FcRn in transgenic (Tg) mice extends the half-life of mouse IgG by reducing its clearance. In this paper, we demonstrate that immunization of these mice with OVA and trinitrophenyl-conjugated human IgG results in a 3- to 10-fold increase of Ag-specific IgM and IgG in serum. The IgM increase was unexpected because FcRn does not bind IgM. Our results showed that the affinity of the Ag-specific IgG was at least as good in Tg mice as in the wild-type (wt) controls, implying appropriate affinity maturation in both groups. Influenza vaccination produced a 2-fold increase in the amount of virus-specific Ab in Tg animals, which proved twice as efficient in a hemagglutination inhibition assay as was the case in wt controls. After immunization, Tg mice displayed significantly larger spleens containing a higher number of Ag-specific B cells and plasma cells, as well as many more granulocytes and dendritic cells, analyzed by ELISPOT and flow cytometric studies. The neutrophils from these Tg mice expressed the Tg FcRn and phagocytosed IgG immune complexes more efficiently than did those from wt mice. These results show that FcRn overexpression not only extends the IgG half-life but also enhances the expansion of Ag-specific B cells and plasma cells. Although both effects increase the level of Ag-specific IgG, the increase in immune response and IgG production seems to be more prominent compared with the reduced IgG clearance.

    Copyright © 2011 by The American Association of Immunologists

  • Analytical Biochemistry, 414 (2011) 117–124 Polyethylene glycol-stabilized lipid disks as model membranes in interaction studies based on electrokinetic capillary chromatography and quartz crystal microbalanceK. Vainikka, K. Reijmar, G. Yohannes, J. Samuelsson, K. Edwards, M. Jussila, M.-L. Riekkola

    Distearoylphosphatidylcholine (DSPC)/cholesterol/distearoylphosphatidylethanolamine (DSPE)–polyethylene glycol 5000 [PEG(5000)] lipid disks, mimicking biological membranes, were used as pseudostationary phase in partial filling electrokinetic capillary chromatography (EKC) to study interactions between pharmaceuticals and lipid disks. Capillaries were coated either noncovalently with a poly(1-vinylpyrrolidone)-based copolymer or covalently with polyacrylamide to mask the negative charges of the fused-silica capillary wall and to minimize interactions between positively charged pharmaceuticals and capillary wall. Although the noncovalent copolymer coating method was faster, better stability of the covalent polyacrylamide coating at physiological pH 7.4 made it more reliable in partial filling EKC studies. Migration times of pharmaceuticals were proportional to the amount of lipids in the pseudostationary phase, and partition coefficients were successfully determined. Because the capillary coatings almost totally suppressed the electroosmotic flow, it was not practical to use the EKC-based method for partition studies involving large molecules with low mobilities. Hence, the applicability of the biomembrane mimicking lipid disks for interactions studies with large molecules was verified by the quartz crystal microbalance technique. Biotinylated lipid disks were then immobilized on streptavidincoated sensor chip surface, and interactions with a high-molecular-mass molecule, lysozyme, were studied. Cryo-transmission electron microscopy and asymmetrical flow field-flow fractionation were used to clarify the sizes of lipid disks used.

    © 2011 Elsevier Inc.

  • Journal of Biological Chemistry, August 12, 2011 Unbinding of hyaluronan accelerates the enzymatic activity of bee hyaluronidaseAttila Iliás, Károly Liliom, Brigitte Greiderer-Kleinlercher, Stephan Reitinger and Günter Lepperdinger

    Hyaluronan (HA), a polymeric glycosaminoglycan ubiquitously present in higher animals is hydrolyzed by hyaluronidases (HAases). Here we used bee HAase as a model enzyme to study HA-HAase interaction. Located in close proximity to the active center, a bulky surface loop, which appears to obstruct one end of the substrate binding groove, was found to be functionally involved in HA turnover. To better apprehend kinetic changes in substrate interaction, binding of high molecular weight HA to catalytically inactive HAase was monitored by means of Quartz Crystal Microbalance technology. Replacement of the delimiting loop by a tetrapeptide interconnection increased the affinity for HA up to 100-fold, with a KD below 1 nM being the highest affinity amongst HA-binding proteins surveyed so far. The experimental data of HA-HAase interaction were further validated showing best fit to the theoretically proposed sequential twosite model. Besides the one, which had been shown previously in course of X-ray structure determination, a previously unrecognized binding site works in conjunction with an unbinding loop that facilitates liberation of hydrolyzed HA.

    © The American Society for Biochemistry and Molecular Biology 2011

  • Analyst 2011, 136, 3777-3782 Collagen I and III and their decorin modified surfaces studied by atomic force microscopy and the elucidation of their affinity toward positive apolipoprotein B-100 residue by quartz crystal microbalanceWitos J, Saint-Guirons J, Meinander K, D'Ulivo L, Riekkola ML

    Collagen, the major component of extracellular matrix (ECM) and the most abundant protein in the human body, is implicated in the development of atherosclerosis. Collagen types I and III were immobilized on fused-silica capillary to investigate their shape, size and structure by atomic force microscopy (AFM). For comparison, collagen was also immobilized on a mica surface. Our studies demonstrated that not only does the adsorption pattern on the substrate vary with the type of collagen, but also the substrate material plays an important role in the fibril formation process. Decorin, which promotes the binding of low-density lipoprotein (LDL) particles with collagen, was investigated for its effect on the fibrillogenesis. On both substrate materials, addition of decorin clearly reduced the fibril diameter of collagen surfaces. Moreover, a quartz crystal microbalance (QCM)-based biosensor approach was applied to clarify and evaluate the affinity of different collagen coatings immobilized on a silicon dioxide sensor chip toward apolipoprotein B-100, the major protein of LDL. The results confirmed the importance of collagen type and their fibrillogenesis on the binding of the positive residues of apolipoprotein B-100 on negatively charged collagen surfaces.

    © Royal Society of Chemistry 2011

  • Innovations in Pharmaceutical Technology 2011, June; 34-40 Improving the Developability of BiopharmaceuticalsZurdo J, Michael R, Stallwood Y, Hedman K and Aastrup T

    Biopharmaceutical development is marred by multiple risks. High levels of attrition during preclinical and clinical development are posing a significant challenge, and pushing drug development costs to levels that are no longer sustainable. Diverse strategies for failing early and cheaply are currently being explored, with an emphasis on translational medicine, predictive technologies, scale-down models and further up-front product characterisation. One of these approaches involves the incorporation of a developability risk assessment – focusing on manufacturability and safety – in the early phases of development, in order to help with the selection and design of products with the right quality attributes. One such attribute is aggregation, which constitutes a significant hurdle for biopharmaceutical development and a potential risk of increased immunogenicity. In this article, we discuss how protein-engineering approaches based on predictive computational methods, together with early analytics, can be utilised to select candidates with enhanced developability. We also provide examples of new orthogonal strategies to assess aggregation and stability at an early stage.

    © 2011 Samedan Ltd

  • Appl Environ Microbiol. 2011 Sep;77(17):6165-71 Magnetosome expression of functional camelid antibody fragments (nanobodies) in Magnetospirillum gryphiswaldensePollithy, Romer, Lang, Müller, Helma, Leonhardt, Rothbauer, Schüler

    Numerous applications of conventional and biogenic magnetic nanoparticles (MNPs), such as in diagnostics, immunomagnetic separations, and magnetic cell labeling, require the immobilization of antibodies. This is usually accomplished by chemical conjugation, which, however, has several disadvantages, such as poor efficiency and the need for coupling chemistry. Here, we describe a novel strategy to display a functional camelid antibody fragment (nanobody) from an alpaca (Lama pacos) on the surface of bacterial biogenic magnetic nanoparticles (magnetosomes). Magnetosome-specific expression of a red fluorescent protein (RFP)-binding nanobody (RBP) in vivo was accomplished by genetic fusion of RBP to the magnetosome protein MamC in the magnetite-synthesizing bacterium Magnetospirillum gryphiswaldense. We demonstrate that isolated magnetosomes expressing MamC-RBP efficiently recognize and bind their antigen in vitro and can be used for immunoprecipitation of RFP-tagged proteins and their interaction partners from cell extracts. In addition, we show that coexpression of monomeric RFP (mRFP or its variant mCherry) and MamC-RBP results in intracellular recognition and magnetosome recruitment of RFP within living bacteria. The intracellular expression of a functional nanobody targeted to a specific bacterial compartment opens new possibilities for in vivo synthesis of MNP-immobilized nanobodies. Moreover, intracellular nanotraps can be generated to manipulate bacterial structures in live cells.

    Copyright © 2011, American Society for Microbiology. All Rights Reserved.

  • Analytical and Bioanalytical Chemistry (2011) 400:1397–1404 Towards a Synthetic Avidin MimicWiklander J, Karlsson BCG, Aastrup T, Nicholls IA

    A series of streptavidin-mimicking molecularly imprinted polymers has been developed and evaluated for their biotin binding characteristics. A combination of molecular dynamics and NMR spectroscopy was used to examine potential polymer systems, in particular with the functional monomers methacrylic acid and 2-acrylamidopyridine. The synthesis of copolymers of ethylene dimethacrylate and one or both of these functional monomers was performed. A combination of radioligand binding studies and surface area analyses demonstrated the presence of selectivity in polymers prepared using methacrylic acid as the functional monomer. This was predicted by the molecular dynamics studies showing the power of this methodology as a prognostic tool for predicting the behavior of molecularly imprinted polymers.

    © Springer-Verlag 2011

  • Analytical Chemistry, 2011, 83, 6040–6046 Three Different Approaches for the Clarification of the Interactions between Lipoproteins and Chondroitin-6-sulfateK. Lipponen, P.W. Stege, G. Cilpa, J. Samuelsson, T. Fornstedt and M.-L. Riekkola

    Two different experimental approaches were used for obtaining a comprehensive view and understanding of the interactions between apolipoprotein B-100 (ApoB-100) of low-density lipoprotein and apolipoprotein E (ApoE) of high-density lipoprotein and chondroitin-6-sulfate (C6S) of arterial proteoglycan. The techniques employed were partial filling affinity capillary electrophoresis (PF-ACE) and continuous flow quartz crystal microbalance (QCM). In addition, molecular dynamic (MD) simulations were used to provide a supportive visual insight into the interaction mechanism. A new tool for analysis of QCM-data was utilized, i.e., adsorption energy distribution calculations, which allowed a deeper understanding of the interactions, especially at different temperatures. The PF-ACE technique probed mainly the strong adsorption interactions whereas in the MD calculations short- and long-range interactions could be distinguished. Although there are differences in the techniques, a pretty good agreement was achieved between the three approaches for the interaction of 19 amino acid peptide of ApoB with C6S giving log affinity constants of 4.66 by QCM, 5.02 by PF-ACE, and 7.39 by MD, and for 15 amino acid peptide of ApoE with C6S 5.34 by QCM, 5.28 by PT-ACE, and 4.60 by MD at physiological temperature 37.0°C.

    © 2011 American Chemical Society

  • Journal of Biological Chemistry (JBC) 2011, May; vol 286:18, 15908-17 The Chondroitin Sulfate A-binding Site of the VAR2CSA Protein Involves Multiple N-terminal DomainsDahlbäck M, Jørgensen LM, Nielsen MA, Clausen TM, Ditlev SB, Resende M, Pinto VV, Arnot DE, Theander TG, Salanti A

    Malaria during pregnancy is a major health problem for African women. The disease is caused by Plasmodium falciparum malaria parasites, which accumulate in the placenta by adhering to chondroitin sulfate A (CSA). The interaction between infected erythrocytes and the placental receptor is mediated by a parasite expressed protein named VAR2CSA. A vaccine protecting pregnant women against placental malaria should induce antibodies inhibiting the interaction between VAR2CSA and CSA. Much effort has been put into defining the part of the 350 kDa VAR2CSA protein that is responsible for binding. It has been shown that full-length recombinant VAR2CSA binds specifically to CSA with high affinity, however to date no sub-fragment of VAR2CSA has been shown to interact with CSA with similar affinity or specificity. In this study, we used a biosensor technology to examine the binding properties of a panel of truncated VAR2CSA proteins. The experiments indicate that the core of the CSA-binding site is situated in three domains, DBL2X-CIDRPAM and a flanking domain, located in the N-terminal part of VAR2CSA. Furthermore, recombinant VAR2CSA subfragments containing this region elicit antibodies with high parasite adhesion blocking activity in animal immunization experiments.

    © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

  • Sensors and Actuators B 153 (2011) 135–144 Systematic investigation of biomolecular interactions using combined frequency and motional resistance measurementsH Anderson, G Wingqvist, T Weissbach, D Wallinder, I Katardjiev, B Ingemarsson

    The resonance frequency of acoustic biosensors is today used as a label-free technique for detecting mass changes on sensor surfaces. In combination with an appropriate continuous flow system it has earlier been used for affinity and kinetic rate determination. Here, we assess the potential of a modified acoustic biosensor, monitoring also the real-time dissipation through the resistance of the sensor, to obtain additional kinetic information related to the structure and conformation of the molecules on the surface. Actual interaction studies, including an attempt to determine avidity, are presented along with thorough verification of the experimental setup utilizing true viscous load exposure together with protein and DNA immobilizations.

    © 2010 Elsevier B.V.

  • Analytical Chemistry (2011) 83: 1000-1007 Photo-Click Immobilization on Quartz Crystal Microbalance Sensors for Selective Carbohydrate-Protein Interaction AnalysesOscar Norberg, Lingquan Deng, Teodor Aastrup, Mingdi Yan and Olof Ramström

    A photoclick method based on azide photoligation and Cu-catalyzed azide-alkyne cycloaddition has been evaluated for the immobilization of carbohydrates to polymeric materials. The biomolecular recognition properties of the materials have been investigated with regard to applicable polymeric substrates and selectivity of protein binding. The method was used to functionalize a range of polymeric surfaces (polystyrene, polyacrylamide, poly(ethylene glycol), poly(2-ethyl-2-oxazoline), and polypropene) with various carbohydrate structures (based on R-D-mannose, β-D-galactose, and N-acetyl-β-D-glucosamine). The functionalized surfaces were evaluated in real-time studies of protein-carbohydrate interactions using a quartz crystal microbalance flow-through system with a series of different carbohydrate-binding proteins (lectins). The method proved to be robust and versatile, resulting in a range of efficient sensors showing high and predictable protein selectivities.

    © 2010 American Chemical Society

  • Protein Engineering, Design & Selection (PEDS) 2010, Nov;23(11):827-34. Epub 2010 Sep 3. The effects of affinity and valency of an albumin-binding domain (ABD) on the half-life of a single-chain diabody-ABD fusion proteinHopp J, Hornig N, Zettlitz KA, Schwarz A, Fuss N, Müller D, Kontermann RE.

    Fusion of small recombinant antibody fragments to an albumin-binding domain (ABD) from streptococcal protein G strongly extends their plasma half-life. This ABD binds with nanomolar affinity to human (HSA) and mouse serum albumin (MSA). It was speculated that an increase in albumin-binding affinity should lead to a further increase in half-life. In the present study, we analyzed the effects of affinity and valency of the ABD on the pharmacokinetic properties of a bispecific single-chain diabody (scDb), applied previously to investigate various half-life extension strategies. The scDb is directed against carcinoembryonic antigen (CEA) and CD3 capable of mediating T cell retargeting to tumor cells. Two scDb derivatives with increased (scDb-ABD-H) and decreased (scDb-ABD-L) affinity as well as an scDb molecule fused to two ABD (scDb-ABD(2)) were generated and produced in mammalian cells. The altered binding of these constructs to HSA and MSA was confirmed by ELISA and quartz crystal microbalance measurements. All constructs bound efficiently to CEA and CD3-positive cells and were able to activate T cells in a target cell-dependent manner, although T cell activation was reduced in the presence of serum albumin. All three derivatives showed a strongly increased half-life in mice as compared with scDb. Compared with the wild-type scDb-ABD, the half-life of scDb-ABD-H exhibited a prolonged half-life and scDb-ABD-L a reduced half-life, while the half-life scDb-ABD(2) was almost identical to that of scDb-ABD. However, these changes were only moderate, indicating that the half-life-extending property of the ABD in mice is only weakly influenced by affinity for serum albumin or valency of albumin binding.

    © Copyright 2010 Oxford University Press

  • August 19, 2013 ATROSAB, a humanized antagonistic anti-tumor necrosis factor receptor one specific antibodyKirstiin A Zettlitz, Verena Lorenz, Peter Scheurich, Klaus Pfizenmaier, Andreas Herrmann & Roland E. Kontermann.
  • Journal of Biological Chemistry (JBC) 2010 Oct 15;285(42):32638-46. Epub 2010 Aug 2 WSS25 inhibits growth of xenografted hepatocellular cancer cells in nude mice by disrupting angiogenesis via blocking bone morphogenetic protein (BMP)/Smad/Id1 signalingQiu H, Yang B, Pei ZC, Zhang Z, Ding K.

    The highly expressed Id1 (inhibitor of DNA binding/differentiation) protein promotes angiogenesis in HCC and is a well established target for anti-angiogenesis therapeutic strategies. Heparan sulfate (HS) mimetics such as PI-88 can abrogate HS-protein interactions to inhibit angiogenesis. Id1 is the direct downstream effector of bone morphogenetic proteins (BMPs), which are angiogenic and HS-binding proteins. Thus, targeting BMPs by HS mimetics may inhibit angiogenesis via attenuating Id1 expression. We report here that a HS mimetic WSS25 potently inhibited the tube formation of HMEC-1 cells on Matrigel and their migration. Meanwhile, WSS25 (25 μg/ml) nearly completely blocked Id1 expression in the HMEC-1 cells as demonstrated by oligo-angiogenesis microarray analysis and further confirmed by RT-PCR and Western blotting. BMP/Smad/Id1 signaling also was blocked by WSS25 treatment in HMEC-1 cells. Importantly, Id1 knockdown in HMEC-1 cells caused the disruption of their tube formation on Matrigel. By employing quartz crystal microbalance analysis, we found that WSS25 strongly bound to BMP2. Moreover, WSS25 impaired BMP2-induced tube formation of HMEC-1 cells on Matrigel and angiogenesis in Matrigel transplanted into C57BL6 mice. Furthermore, WSS25 (100 mg/kg) abrogated the growth of HCC cells xenografted in male nude mice. Immunohistochemical analysis showed that both the expression of Id1 and the endothelial cell marker CD31 were lower in the WSS25-treated tumor tissue than in the control. Therefore, WSS25 is a potential drug candidate for HCC therapy as a tumor angiogenesis inhibitor.

    © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

  • Malaria Journal 2010, 9:100 The kinetics of antibody binding to Plasmodium falciparum VAR2CSA PfEMP1 antigen and modeling of PfEMP1 antigen packing on the membrane knobsLars M Jørgensen, Ali Salanti, Tina Dobrilovic, Lea Barfod, Tue Hassenkam, Thor G Theander, Lars Hviid and David E Arnot

    Infected humans make protective antibody responses to the PfEMP1 adhesion antigens exported by Plasmodium falciparum parasites to the erythrocyte membrane, but little is known about the kinetics of this antibody-receptor binding reaction or how the topology of PfEMP1 on the parasitized erythrocyte membrane influences antibody association with, and dissociation from, its antigenic target.

    © 2010 Jørgensen et al; licensee BioMed Central Ltd.

  • Molecular Biotechnology (2010) 46:265–278 Humanization of a Mouse Monoclonal Antibody Directed Against a Cell Surface-Exposed Epitope of Membrane-Associated Heat Shock Protein 70 (Hsp70)Kirstin A. Zettlitz, Julia Seitter, Dafne Müller, Roland E. Kontermann

    The translocation of heat shock protein 70 (mHsp70) into the plasma membrane has been found to be associated with various cancers including breast cancer, head-and-neck cancer, and acute myeloid leukemia. Parts of the C-terminal substrate-binding domain (SBD) of mHsp70 are accessible to binding by monoclonal antibodies (mAb). One of these mAbs, cmHsp70.1, has been extensively studied and showed promising results as diagnostic and therapeutic antibody. Here, we describe cloning and humanization of cmHsp70.1 by complementarity determining region grafting resulting in an antibody (humex) possessing a similar affinity (3 nM) as the parental antibody and an improved production and thermal stability. Epitope mapping confirmed that the parental, chimeric, and humanized antibodies recognize the same region including amino acids 473–504 of the SBD. Hence, this humanized antibody provides a basis for further development of an anti-mHsp70 antibody therapy.

    © Springer Science+Business Media, LLC 2010

  • THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 30, pp. 26616 –26627 Heparin Impairs Angiogenesis through Inhibition of MicroRNA-10bXiaokun Shen, Jianping Fang, Xiaofen Lv, Zhicao Pei, Ying Wang, Songshan Jiang, and Kan Ding

    Heparin, which has been used as an anticoagulant drug for decades, inhibits angiogenesis, whereas thrombin promotes tumor-associated angiogenesis. However, the mechanisms underlying the regulation of angiogenesis by heparin and thrombin are not well understood. Here, we show that microRNA-10b (miR-10b) is down-regulated by heparin and up-regulated by thrombin in human microvascular endothelial cells (HMEC-1). Overexpression of miR-10b induces HMEC-1 cell migration, tube formation, and angiogenesis, and downregulates homeobox D10 (HoxD10) expression via direct binding of miR-10b to the putative 3 UTR of HoxD10. In addition, HMEC-1 cell migration and tube formation are induced by HoxD10 knockdown, whereas angiogenesis is arrested when HoxD10 expression is increased after anti-miR-10b or heparin treatments. Furthermore, expression of miR-10b and its transcription factor Twist are up-regulated by thrombin, whereas

    HoxD10 expression is impaired by thrombin. Using quartz crystal microbalance analysis, we show that heparin binds to thrombin, thereby inhibiting thrombin-induced expression of Twist

    and miR-10b. However, the expression of miR-10b is not attenuated by heparin any more after thrombin expression is silenced by its siRNA. Interestingly, we find that heparin attenuates miR-10b expression and induces HoxD10 expression in vivo to inhibit angiogenesis and impair the growth of MDA-MB-231 tumor xenografts. These results provide insight into the molecular

    mechanism by which heparin and thrombin regulate angiogenesis.

    © 2011 by The American Society for Biochemistry and Molecular Biology, Inc

  • Chemical Communications, Vol. 46, Issue 14, 2441-2443, 2010 Dynamic glycovesicle systems for amplified QCM detection of carbohydrate-lectin multivalent biorecognitionEugene Mahon, Teodor Aastrup and Mihail Barboiu

    We describe multivalent biorecognition of adsorbed lectin layers by biomimetic sensing nanoplatforms based on dynamic glycovesicles in a continuous flow QCM setup.

    © The Royal Society of Chemistry 2010

  • Analytical Bioanalytical Chemistry (2010) 396: 1373-1380 Quartz crystal microbalance, a valuable tool for elucidation of interactions between apoB-100 peptides and extracellular matrix componentsLucia D’Ulivo, Julien Saint-Guirons, Björn Ingemarsson and Marja-Liisa Riekkola

    Atherosclerosis has received wide attention as a primary cause of premature death in developed countries. The retention of low-density lipoprotein (LDL) particles in the intima, the inner layer of the capillaries, has been imputed as the main cause of the development of atherosclerotic plaques. The entrapment of LDL is mainly due to the specific interaction between the lysine-rich site on apolipoprotein B-100 (apoB-100), a major apolipoprotein of LDL, and extracellular matrix (ECM) components such as collagen, proteoglycans, and glycosaminoglycans (GAGs). Although valuable techniques already exist for studies on apoB-100 and ECM interactions, there is continued need for miniaturized tools that can complement the tools already available and even provide totally new data. This work explores the applicability of the quartz crystal microbalance (QCM) for interaction studies between apoB-100 peptide fragments and various components of the ECM. Two positive peptide fragments, PP and PP2, and two components of the ECM, collagen I and a selected GAG, chondroitin 6-sulfate (C6S), were immobilized on polystyrene and carboxyl sensor chips. C6S was injected as analyte for PP- and PP2-coated surfaces, while PP was the analyte for collagen I and C6S surfaces. The estimated dissociation constant (KD) indicates that the interactions occur via the positive residues, lysine and arginine, of apoB-100. The continuous-flow QCM system employed in this study is shown to be an excellent tool for the elucidation of interactions between these types of biomolecules.

    © Springer-Verlag 2009

  • Analytical Biochemistry vol. 398, Issue 2, 15 March 2010, Pages 161-168 Optimizing immobilization on two-dimensional carboxyl surface: pH dependence of antibody orientation and antigen binding capacityZhichao Pei, Henrik Anderson, Annica Myrskog, Gunnar Dunér, Björn Ingemarsson and Teodor Aastrup

    The performance of immunosensors is highly dependent on the amount of immobilized antibodies and their remaining antigen binding capacity. In this work, a method for immobilization of antibodies on a two-dimensional carboxyl surface has been optimized using quartz crystal microbalance biosensors. We show that successful immobilization is highly dependent on surface pKa, antibody pI, and pH of immobilization buffer. By the use of EDC/sulfo-NHS (1-ethyl 3-[3-dimethylaminopropyl] carbodiimide hydrochloride/N-hydroxysulfosuccinimide) activation reagents, the effect of the intrinsic surface pKa is avoided and immobilization at very low pH is therefore possible, and this is important for immobilization of acidic proteins. Antigen binding capacity as a function of immobilization pH was studied. In most cases, the antigen binding capacity followed the immobilization response. However, the antigen-to-antibody binding ratio differed between the antibodies investigated, and for one of the antibodies the antigen binding capacity was significantly lower than expected from immobilization in a certain pH range. Tests with anti-Fc and anti-Fab2 antibodies on different antibody surfaces indicated that the orientation of the antibodies on the surface had a profound effect on the antigen binding capacity of the immobilized antibodies.

    © 2009 Elsevier Inc.

  • Chemical Communications, Vol 46, Issue: 30, 5491-5493, 2010 Multivalent recognition of lectins by glyconanoparticle systemsMahon E, Aastrup T, Barboiu M

    Multivalent recognition of lectin layers by glyconanoparticle sugar-clusters has been used to study the carbohydrate-protein interactions in a QCM sensing setup.

    © The Royal Society of Chemistry 2010

  • Nature Structural & Molecular Biology vol. 17, no. 1, January 2010, (13 December, 2009) Modulation of protein properties in living cells using nanobodiesAxel Kirchhofer, Jonas Helma, Katrin Schmidthals, Carina Frauer, Sheng Cui, Annette Karcher, Mireille Pellis, Serge Muyldermans, Corella S. Casas-Delucchi, M. Cristina Cardoso, Heinrich Leonhardt, Karl-Peter P. Hopfner and Ulrich Rothbauer

    Protein conformation is critically linked to function and often controlled by interactions with regulatory factors. Here we report the selection of camelid-derived single-domain antibodies (nanobodies) that modulate the conformation and spectral properties of the green fluorescent protein (GFP). One nanobody could reversibly reduce GFP fluorescence by a factor of 5, whereas its displacement by a second nanobody caused an increase by a factor of 10. Structural analysis of GFP-nanobody complexes revealed that the two nanobodies induce subtle opposing changes in the chromophore environment, leading to altered absorption properties. Unlike conventional antibodies, the small, stable nanobodies are functional in living cells. Nanobody-induced changes were detected by ratio imaging and used to monitor protein expression and subcellular localization as well as translocation events such as the tamoxifen-induced nuclear localization of estrogen receptor. This work demonstrates that protein conformations can be manipulated and studied with nanobodies in living cells.

    © 2010 Nature America, Inc.

  • Innovations in Pharmaceutical Technology - Issue 29(2009) (Online) Crude Sample Analysis Made EasyAlexander Kovacs

    A new system enables biomolecular interaction analyses to be performed on impure samples – thereby saving on time, labour and costs. Knowing the kinetic properties of biomolecules is increasingly important in drug research. Previously, it has been necessary to obtain purified molecules for such studies as impure samples pose challenges to non-specific binding and microfluidics. The ability to analyse impure samples would thus provide the benefits of saving time, labour and cost. At Attana, we have developed a system that makes this possible. In this article, we review the system and describe how it can be used in the screening of antibodies and determining their offrates in serum containing hybridoma supernatants.

    IPTonline © 2009 The Pharmaceutical Technology Journal

  • Bioconjugate Chemistry, Vol. 20, Issue 12, 2364-2370, Dec. 2009 Photo-Click Immobilization of Carbohydrates on Polymeric Surfaces-A Quick Method to Functionalize Surfaces for Biomolecular Recognition StudiesOscar Norberg, Lingquan Deng, Mingdi Yan and Olof Ramström

    Methods to rapidly functionalize specific polymeric surfaces with alkynes, which can subsequently be linked to azide-containing carbohydrates, are presented. The methods comprise two main concepts: azide photoligation and Cu-catalyzed azide-alkyne cycloaddition. 2-Azidoethyl-functionalized alpha-d-mannopyranoside was synthesized and covalently attached to alkyne-functionalized polymeric surfaces using the techniques. The protein recognition properties of the carbohydrate-presenting surfaces were evaluated using quartz crystal microbalance biosensor instrumentation.

    © 2009 American Chemical Society

  • The Journal of Biological Chemistry (2009) 284, 25612-25619 Biodistribution of a Bispecific Single-chain Diabody and Its Half-life Extended DerivativesRoland Stork, Emmanuelle Campigna, Bruno Robert, Dafne Müller and Roland E. Kontermann

    Small recombinant antibody molecules such as bispecific single-chain diabodies (scDb) possessing a molecular mass of approximately 55 kDa are rapidly cleared from circulation. We have recently extended the plasma half-life of scDb applying various strategies including PEGylation, N-glycosylation and fusion to an albumin-binding domain (ABD) from streptococcal protein G. Here, we further analyzed the influence of these modifications on the biodistribution of a scDb directed against carcinoembryonic antigen (CEA) and CD3 capable of retargeting T cells to CEA-expressing tumor cells. We show that a prolonged circulation time results in an increased accumulation in CEA+ tumors, which was most pronounced for scDb-ABD and PEGylated scDb. Interestingly, tumor accumulation of the scDb-ABD fusion protein was approximately 2-fold higher compared with PEGylated scDb, although both molecules exhibit similar plasma half-lives and similar affinities for CEA. Comparing half-lives in neonatal Fc receptor (FcRn) wild-type and FcRn heavy chain knock-out mice the contribution of the FcRn to the long plasma half-life of scDb-ABD was confirmed. The half-life of scDb-ABD was approximately 2-fold lower in the knock-out mice, while no differences were observed for PEGylated scDb. Binding of the scDb derivatives to target and effector cells was not or only marginally affected by the modifications, although, compared with scDb, a reduced cytotoxic activity was observed for scDb-ABD, which was further reduced in the presence of albumin. In summary, these findings demonstrate that the extended half-life of a bispecific scDb translates into improved accumulation in antigen-positive tumors but that modifications might also affect scDb-mediated cytotoxicity.

    © 2009 by American Society for Biochemistry and Molecular Biology

  • Biosensors and Bioelectronics 24 (2009) 3387–3390 On the applicability of high frequency acoustic shear mode biosensing in view of thickness limitations set by the film resonanceG. Wingqvist, H. Anderson, C. Lennartsson, T. Weissbach, V. Yantchev, A. Lloyd Spetz

    The IC-compatible thin film bulk acoustic resonator (FBAR) technology has made it possible to move the thickness excited shear mode sensing of biological layers into a new sensing regime using substantially higher operation frequencies than the conventionally used quartz crystal microbalance (QCM). The limitations of the linear range set by the film resonance using viscoelastic protein films are here for the first time addressed specifically for FBARs operating at 700MHz up to 1.5 GHz. Two types of protein multilayer sensing were employed; one utilizing alternating layers of streptavidin and biotinated BSA and the other using stepwise cross-linking of fibrinogen with EDC/NHS activation of its carboxyl groups. In both cases the number of protein layers within the linear regime is well above the number of protein layers usually used in biosensor applications, further verifying the applicability of the FBAR as a biosensor. Theoretical calculations are also presented using wel established physical models to illustrate the expected behavior of the FBAR sensor, in view of both the frequency and the dissipation shifts.

    © 2009 Elsevier B.V.

  • PNAS vol. 106, no. 12, 4623–4628 (Mar. 2009) Controlled release of functional proteins through designer self-assembling peptide nanofiber hydrogel scaffoldSotirios Koutsopoulos, Larry D. Unsworth, Yusuke Nagai, and Shuguang Zhang

    The release kinetics for a variety of proteins of a wide range of molecular mass, hydrodynamic radii, and isoelectric points through a nanofiber hydrogel scaffold consisting of designer self-assembling peptides were studied by using single-molecule fluorescence correlation spectroscopy (FCS). In contrast to classical diffusion experiments, the single-molecule approach allowed for the direct determination of diffusion coefficients for lysozyme, trypsin inhibitor, BSA, and IgG both inside the hydrogel and after being released into the solution. The results of the FCS analyses and the calculated pristine in-gel diffusion coefficients were compared with the values obtained from the Stokes–Einstein equation, Fickian diffusion models, and the literature. The release kinetics suggested that protein diffusion through nanofiber hydrogels depended primarily on the size of the protein. Protein diffusivities decreased, with increasing hydrogel nanofiber density providing a means of controlling the release kinetics. Secondary and tertiary structure analyses and biological assays of the released proteins showed that encapsulation and release did not affect the protein conformation and functionality. Our results show that this biocompatible and injectable designer self-assembling peptide hydrogel system may be useful as a carrier for therapeutic proteins for sustained release applications.

    © 2009 PNAS by the National Academy of Sciences

  • Tumor Biology 30:26–36 (Feb. 2009) Characterization of Monoclonal Antibodies Directed against Squamous Cell Carcinoma Antigens: Report of the Second TD-10 WorkshopK. Nustad, O. Nilsson, K. Majnesjö, A. Murakami, N. Sugino, D.J. Warren, and H. Kato

    Eight monoclonal antibodies directed against Squamous Cell Carcinoma Antigens (A1 and A2) were collected and evaluated by three working groups. Recombinant antigens, fusion proteins and native antigens from normal tissue were used to evaluate antibody specificity. Five antibodies reacted with both A1 and A2. Two of these antibodies (K123 and K131) showed related binding characteristics, whereas SCC140, K182 and SCC111 demonstrated unique epitope specificity and were not related to the reference antibodies included (F1H3, F2H7 and SCC107). SCC111 reacted particularly well with antigen on Western blot, indicating that the epitope was partly hidden when the antigen was in solution. Two antibodies (SCC103 and SCC109) reacted only with A2 and the fusion protein A1/A2, indicating that they recognized an A2 epitope in exon 8. The A2-specific antibodies are unique in their binding to A2 and are different from the reference antibodies included (SCC104 and K122). SCC103 is probably the best A2-specific antibody available. One antibody, K136, was A1-specific and is related to reference antibody K135. The new antibodies can be used to establish immunometric assays for specific measurement of A1, A2 or both A1 and A2 together.

    © 2009 S. Karger AG, Basel

  • Langmuir, 2008, 24 (14), pp 7559–7564 Surface-Confined Photopolymerization of pH-Responsive Acrylamide/Acrylate-Brushes on Polymer Thin FilmsGunnar Dunér, Henrik Anderson, Annica Myrskog, Maria Hedlund, Teodor Aastrup, Olof Ramström

    Dynamic acrylamide/acrylate polymeric brushes were synthesized at gold-plated quartz crystal surfaces. The crystals were initially coated with polystyrene-type thin films, derivatized with photolabile iniferter groups, and subsequently subjected to photoinitiated polymerization in acrylamide/acrylate monomer feeds. This surface-confined polymerization method enabled direct photocontrol over the polymerization, as followed by increased frequency responses of the crystal oscillations in a quartz crystal microbalance (QCM) instrumentation. The produced polymer layers were also found to be highly sensitive to external acid/base stimuli. Large oscillation frequency shifts were detected when the brushes were exposed to buffer solutions of different pH. The dynamic behavior of the resulting polymeric brushes was evaluated and the extent of expansion and contraction of the films monitored by the QCM setup in situ in real-time. The resulting responses were rapid and the effects were fully reversible. Low pH resulted in full contractions of the films, whereas higher pH yielded maximal expansion in order to minimize repulsion around the charged acrylate centers. The surfaces proved also highly robust, since the responsiveness was reproducible over many cycles of repeated expansion and contraction.

    © 2008 American Chemical Society

  • Journal of Molecular Biology (2008) 384, 1143–1156 Generation, Affinity Maturation, and Characterization of a Human Anti-Human NKG2D Monoclonal Antibody with Dual Antagonistic and Agonistic ActivityKa Yin Kwong, Sivasubramanian Baskar, Hua Zhang, Crystal L. Mackall and Christoph Rader

    In humans,NKG2Dis an activating receptor on natural killer (NK) cells and a costimulatory receptor on certain Tcells and plays a central role in mediating immune responses in autoimmune diseases, infectious diseases, and cancer. Monoclonal antibodies that antagonize or agonize immune responses mediated by human NKG2D are considered to be of broad and potent therapeutic utility. Nonetheless, monoclonal antibodies to NKG2D that are suitable for clinical investigations have not been published yet. Here, we describe the generation, affinity maturation, and characterization of a fully human monoclonal antibody to human NKG2D. Using phage display technology based on a newly generated naïve human Fab library in phage display vector pC3C followed by a tandem chain shuffling process designed for minimal deviation from natural human antibody sequences,we selected a human Fab, designated KYK-2.0, with high specificity and affinity to human NKG2D. KYK-2.0 Fab blocked the binding of the natural human NKG2D ligands MICA, MICB, and ULBP2 as potently as a commercially available mouse anti-human NKG2D monoclonal antibody in immunoglobulin G (IgG) format. Conversion of KYK-2.0 Fab to IgG1 resulted in subnanomolar avidity for human NKG2D. KYK-2.0 IgG1 was found to selectively recognize defined subpopulations of human lymphocytes known to express NKG2D, that is, the majority of human CD8+, CD16+, and CD56+ cells as well as a small fraction of human CD4+ cells. In solution, KYK-2.0 IgG1 interfered with the cytolytic activity of ex vivo expanded human NK cells. By contrast, immobilized KYK-2.0 IgG1 was found to strongly induce human NK cell activation. The dual antagonistic and agonistic activity promises a wide range of therapeutic applications for KYK-2.0 IgG1 and its derivatives.

    © 2008 Published by Elsevier Inc.

  • Talanta 77 (2008) 468–472 Flow-injection assay of the pathogenic bacteria using lectin-based quartz crystal microbalance biosensorGulnara Safina, Margret van Lier, Bengt Danielsson

    A novel flow-injection assay of the pathogenic enterobacteria using novel lectin-based quartz crystal microbalance (QCM) biosensor has been proposed. The biosensing part of the analytical device contained the lectins – Concanavalin A, lectins from Ulex europeus, Maackia amurensis, Lens culinaris, wheat germ agglutinin – immobilized on the gold surface of quartz crystal electrode which served as a transducer. The immobilization of lectins was carried out using amine coupling on the surface of the crystal modified with 11-mercaproundecanoic acid. The biosensor makes it possible to identify the presence of different bacterial using the lectins immobilized on the surface of QCM crystal which bind specifically to the certain oligosaccharides present on the cell wall of the bacteria injected. The working conditions of the biosensor – pH of buffer solutions, concentration of the immobilized lectins, dilution of the bacterial cells, regeneration solution and flow rate –were optimized. The use of solution of glycine (pH 2.5) makes it possible to remove the formed complex from the crystal surface to make it reusable and ready for the next experiment. The proposed biosensor is able to detect 10³ cells. The flow-injection assay of the bacterial cells takes about 30 min.

    © 2008 Published by Elsevier B.V.

  • Analytical Chemistry 79 (18): 6897-6902 (Sep. 15, 2007) Photoderivatized Polymer Thin Films at Quartz Crystal Microbalance Surfaces: Sensors for Carbohydrate-Protein InteractionsYuxin Pei, Hui Yu, Zhichao Pei, Matthias Theurer, Carolin Ammer, Sabine André, Hans-Joachim Gabius, Mingdi Yan and Olof Ramström

    Photoderivatized polymer-coated gold surfaces have been developed following a perfluorophenylazide-based double ligation strategy. Gold-plated quartz crystal microbalance (QCM) crystals were initially covalently functionalized with a monolayer of poly(ethylene glycol) (PEG), using photo- or thermolytic nitrene formation and insertion. The polymer surfaces were subsequently used as substrates for photoinsertion of carbohydrate-derivatized photoprobes, yielding different recognition motifs for selective protein binding. The resulting robust and biocompatible sensor surfaces were applied to a flow-through QCM instrument for monitoring lectin-carbohydrate interactions in real time. The results clearly show the predicted lectin selectivity, demonstrating the applicability of the approach.

    © 2007 American Chemical Society. All rights reserved.

  • Langmuir 2007, 23, 11147-11156 Mode of Heavy Meromyosin Adsorption and Motor Function Correlated with Surface Hydrophobicity and ChargeNuria Albet-Torres, John O’Mahony, Christy Charlton, Martina Balaz, Patricia Lisboa, Teodor Aastrup, Alf Månsson and Ian A. Nicholls

    The in vitro motility assay is valuable for fundamental studies of actomyosin function and has recently been combined with nanostructuring techniques for the development of nanotechnological applications. However, the limited understanding of the interaction mechanisms between myosin motor fragments (heavy meromyosin, HMM) and artificial surfaces hampers the development as well as the interpretation of fundamental studies. Here we elucidate the HMM-surface interaction mechanisms for a range of negatively charged surfaces (silanized glass and SiO2), which is relevant both to nanotechnology and fundamental studies. The results show that the HMM-propelled actin filament sliding speed (after a single injection of HMM, 120 μg/mL) increased with the contact angle of the surfaces (in the range of 20-80°). However, quartz crystal microbalance (QCM) studies suggested a reduction in the adsorption of HMM (with coupled water) under these conditions. This result and actin filament binding data, together with previous measurements of the HMM density (Sundberg, M.; Balaz, M.; Bunk, R.; Rosengren-Holmberg, J. P.; Montelius, L.; Nicholls, I. A.; Omling, P.; Tågerud, S.; Månsson, A. Langmuir 2006, 22, 7302-7312. Balaz, M.; Sundberg, M.; Persson, M.; Kvassman, J.; Månsson, A. Biochemistry 2007, 46, 7233-7251), are consistent with (1) an HMM monolayer and (2) different HMM configurations at different contact angles of the surface. More specifically, the QCM and in vitro motility assay data are consistent with a model where the molecules are adsorbed either via their flexible C-terminal tail part (HMMC) or via their positively charged N-terminal motor domain (HMMN) without other surface contact points. Measurements of ú potentials suggest that an increased contact angle is correlated with a reduced negative charge of the surfaces. As a consequence, the HMMC configuration would be the dominant configuration at high contact angles but would be supplemented with electrostatically adsorbed HMM molecules (HMMN configuration) at low contact angles. This would explain the higher initial HMM adsorption (from probability arguments) under the latter conditions. Furthermore, because the HMMN mode would have no actin binding it would also account for the lower sliding velocity at low contact angles. The results are compared to previous studies of the microtubule-kinesin system and are also discussed in relation to fundamental studies of actomyosin and nanotechnological developments and applications.

    © 2007 American Chemical Society

  • Journal of Colloid and Interface Science (2007), doi:10.1016/j.jcis.2007.05.016 Forces and friction between hydrophilic and hydrophobic surfaces: Influence of oleate speciesKatarina Theander, Robert J. Pugh, and Mark W. Rutland

    The atomic force microscope has been used to investigate normal surface forces and lateral friction forces at different concentrations of sodium oleate, a frequently used fatty acid in the deinking process. The measurements have been performed using the colloidal probe technique with bead materials consisting of cellulose and silica. Cellulose was used together with a printing ink alkyd resin and mica, whereas silica was used with a hydrophobized silica wafer. The cellulose–alkyd resin system showed stronger double layer repulsion and the friction was reduced with increasing surfactant concentration. The adhesive interaction disappeared immediately on addition of sodium oleate. The normal surface forces for cellulose–mica indicated no apparent adsorption of the sodium oleate however, the friction coefficient increased on addition of sodium oleate, which we ascribe to some limited adsorption increasing the effective surface roughness. The silica–hydrophobic silica system showed a completely different surface force behavior at the different concentrations. An attractive hydrophobic interaction was evident since the surfaces jumped into adhesive contact at a longer distance than the van der Waals forces would predict. The strong adhesion was reflected in the friction forces as a nonlinear relationship between load and friction and a large friction response at zero applied load. Indirect evidence of adsorption to the hydrophilic silica surface was also observed in this case, and QCM studies were performed to confirm the adsorption of material to both surfaces.

    © 2007 Published by Elsevier Inc.

  • Review of Scientific Instruments 78, 083110 (2007) System for in situ studies of atmospheric corrosion of metal films using soft x-ray spectroscopy and quartz crystal microbalanceJ. Forsberg, L.-C. Duda, A. Olsson, T. Schmitt, J. Andersson, J. Nordgren, J. Hedberg, C. Leygraf, T. Aastrup, D. Wallinder, and J.-H. Guo

    We present a versatile chamber (“atmospheric corrosion cell”) for soft x-ray absorption/emission spectroscopy of metal surfaces in a corrosive atmosphere allowing novel in situ electronic structure studies. Synchrotron x rays passing through a thin window separating the corrosion cell interior from a beamline vacuum chamber probe a metal film deposited on a quartz crystal microbalance (QCM) or on the inside of the window.We present some initial results on chloride induced corrosion of iron surfaces in humidified synthetic air. By simultaneous recording of QCM signal and soft x-ray emission from the corroding sample, correlation between mass changes and variations in spectral features is facilitated.

    © 2007 American Institute of Physics.

  • 2007 Flow Injection Assay of the Path ogenic Bacteria Using Lectin-based QCM BiosensorGulnara Safina, Margret van Lier and Bengt Danielsson, Pure and Applied Biochemistry, Chemical Center.

    A flow injection assay for the detection and identification of the pathogenic bacteria strains using lectin-based QCM biosensor has been proposed

  • Sensors and Actuators B 123 (2007) 21–26 Quartz crystal microbalance biosensor design II. Simulation of sample transportMats Jönsson, Henrik Anderson, Ulf Lindberg, and Teodor Aastrup

    The influence of flow cell geometry on sample dispersion in a quartz crystal microbalance (QCM) biosensor system was investigated. A circular and a rectangular flow cell and corresponding sensor electrodes were studied experimentally and modelled using a coupled Navier–Stokes and convection–diffusion model. Finite element simulations showed that dispersion phenomena in a flow cell can be significantly reduced with the rectangular flow cell compared to a circular system. Experimental results from measurement of the time-dependent viscosity change of a model sample indicate that the sample delivery system has a predominant effect on the dispersion of the whole sensor system. Consequently, improvement of the sensor flow cell should be accompanied with improvement of the sample delivery system. With reference to kinetic studies of biological interactions, the current dispersion should have little effect on the results for studies of interaction pairs with relatively slow to normal binding rates such as antibody–antigen interactions. Incentive for further development of the flow cell and sample delivery system exists primarily for applications with high reaction rates such as for certain receptor ligand interactions.

    © 2007 Elsevier B.V. All rights reserved.

  • Sensors and Actuators B 123 (2007) 27–34 Quartz crystal microbalance sensor design I. Experimental study of sensor response and performanceHenrik Anderson, Mats Jönsson, Lars Vestling, Ulf Lindberg, and Teodor Aastrup

    This paper investigates a novel quartz crystal microbalance (QCM) biosensor with a small and rectangular flow cell along with a correspondingly shaped crystal electrode. The sensor was evaluated with impedance analysis and compared to standard circular sensor crystals and sensor crystals with small circular electrodes. Comparative QCM measurements on an antibody–antigen interaction system were carried out on the rectangular and standard circular sensor systems. Impedance analysis and subsequent data extraction of the three different sensor crystals showed that the smaller sensors had significantly higher Q-values in air, but that liquid load on the electrodes lowered the Q-values radically for all crystals. Under liquid load, Q-values for the standard circular and the rectangular sensors were similar whereas the Q-value for the small circular sensor was 50% higher. QCM experiments showed that the QCM system with rectangular crystal electrodes was fully functional in a liquid environment. The rectangular system showed higher and more rapid responses for series of antibody injections, albeit at a higher noise level than the standard system. The study elucidates a significant potential for improvement of sensor performance by optimising the sensor electrode size and shape together with the flow cell geometry.

    © 2007 Elsevier B.V. All rights reserved.

  • Biosensors and Bioelectronics 22 (2006) 42–48 Quartz crystal microbalance bioaffinity sensor for rapid identification of glycosyldisulfide lectin inhibitors from a dynamic combinatorial libraryZhichao Pei, Rikard Larsson, Teodor Aastrup, Henrik Anderson, Jean-Marie Lehn and Olof Ramström

    Carbohydrate–lectin interactions were probed with dynamic combinatorial libraries, using the plant lectin Concanavalin A as target species. The dynamic combinatorial libraries were generated from a pool of thiol components through reversible thiol–disulfide interchange, and screened using a simple and efficient method based on a quartz crystal microbalance setup. It was found that dimers based on 1-thio- and 6-thio-mannose analogues were the most active inhibitors. Furthermore, the results clearly show that the 6-thio-mannose possess unique characteristics compared to its oxygen-containing counterpart.

    © 2005 Elsevier B.V. All rights reserved.

  • Bioorganic & Medicinal Chemistry Letters 15 (2005) 2707–2710 Redox-responsive and calcium-dependent switching of glycosyldisulfide interactions with Concanavalin AZhichao Pei, Teodor Aastrup, Henrik Anderson and Olof Ramström

    Glycosyldisulfides can interact efficiently with carbohydrate-binding entities. This has been shown for a range of thiosaccharide dimers when tested for their effects against the lectin Concanavalin A using a modified quartz crystal microbalance-technique. Contrary to the thiosaccharide monomers, showing no significant binding up to 10 mM, several of the dimers showed IC50-values in the low millimolar range. Three of the glycosyldisulfides tested also displayed very high positive apparent cooperativity effects that were found to be both calcium-dependent and redox-responsive.

    © 2005 Elsevier Ltd. All rights reserved.

  • Biosensors and Bioelectronics 21 (2005) 60–66 Study of real-time lectin–carbohydrate interactions on the surface of a quartz crystal microbalanceZhichao Pei, Henrik Anderson, Teodor Aastrup and Olof Ramström

    A quartz crystal microbalance (QCM) biosensor system for lectin–carbohydrate interactions has been developed. Yeast mannan was immobilised on polystyrene-coated quartz crystals, and interactions tested with the lectin Concanavalin A (Con A). The biosensor could be easily operated, where mannan immobilisation and all binding analyses were performed in real-time using a flow-through system. The apparent binding constant for yeast mannan to Con A was estimated to be 0.4 _M, well in accordance to reported literature values. In addition, the effective concentration values (EC50-values) for a series of mannose/mannoside ligands, acting as competitors to the mannan/Con A interaction, were determined to range from 0.18 to 5.3 mM, in good correlation with a related enzyme-labeled lectin assay (ELLA) protocol.

    © 2004 Elsevier B.V. All rights reserved.

  • Analytical Biochemistry 341 (2005) 89-93 Electro-immobilization of proinsulin C-peptide to a quartz crystal microbalance sensor chip for protein affinity purificationErmias Melles, Henrik Anderson, Daniel Wallinder, Jawed Shafqat, Tomas Bergman, Teodor Aastrup and Hans Jörnvall

    Proinsulin C-peptide was electro-immobilized to a quartz crystal microbalance sensor chip, localizing this low-pI peptide for covalent attachment to activated surface carboxyl groups. The resulting chip was used in a continuous flow biosensor to capture anti-C-peptide antibodies, which could subsequently be eluted in 5% formic acid between air bubbles for efficient recovery and mass spectrometric identification. The method is reproducible through repeated cycles, providing affinity purification of proteins under real-time monitoring of the binding and elution processes.

    © 2005 Elsevier Ltd. All rights reserved.

  • Protein Engineering, Design & Selection (2005) 18(11):537-546 Isolation of novel single-chain Cro proteins targeted for binding to the bcl-2 transcription initiation site by repertoire selection and subunit combinatoricsKristina Jonas, Erhard Van Der Vries, Mikael T.I.Nilsson and Mikael Widersten

    New designed DNA-binding proteins may be recruited to act as transcriptional regulators and could provide new therapeutic agents in the treatment of genetic disorders such as cancer. We have isolated tailored DNA-binding proteins elected for affinity to a region spanning the transcription initiation site of the human bcl-2 gene. The proteins were derived from a single-chain derivative of the lambda Cro protein (scCro), randomly mutated in its recognition helices to construct libraries of protein variants of distinct DNA binding properties. By phage display-afforded affinity selections combined with recombination of shuffled subunits, protein variants were isolated, which displayed high affinity for the target bcl-2 sequence, as determined by electrophoretic mobility shift and biosensor assays. The proteins analyzed were moderately sequence-specific but provide a starting point for further maturation of desired function.

    © 2005 Oxford Journals. All rights reserved.

  • Analytica Chimica Acta 536 (2005) 191–196 Molecularly imprinted polymer thin films on quartz crystal microbalance using a surface bound photo-radical initiatorTheeraphon Piacham, Åsa Josell, Hans Arwin, Virapong Prachayasittikul, Lei Ye

    The present work aims to study preparation of thin MIP films on a gold-coated QCM resonator in a well-controlled and reproducible manner. A surface-bound photo-radical initiator was used to furnish an imprinting polymerization in a dilute solution of template, functional monomer and crosslinker. Prior to polymerization, the photo-initiator was covalently coupled to a self-assembled monolayer of carboxyl terminated alkanethiol on a gold surface. With this approach we were able to control the thickness of the MIP film to be below 50nm, where the selective recognition of target analytes can be easily detected by the underlying quartz crystal resonator. When used in a flow injection analysis system, the assembled QCM sensor generated a large frequency change (>30 Hz) upon encountering a small amount of analyte (0.19 mM). The sensor had a very short response time (<1 min), and displayed certain chiral selectivity towards the original template, (S)-propranolol at a concentration higher than 0.38mM in aqueous solution.

    © 2004 Elsevier B.V. All rights reserved.

  • Kinetic and Affinity Characterization – Monoclonal Antibodies

    Determine on/off rates and equilibrium dissociation constants of myoglobin–antimyoglobin interactions

  • Kinetic and Affinity Characterization – Polyclonal Antibodies

    To determine the kinetics of the interaction between two different polyclonal antibodies and
    their respective peptide and protein antigen

  • Epitope mapping

    Development of assay for studies of PSA monoclonal antibodies’ binding characteristics


  • Active Concentration Determination

    To determine the active concentration of myoglobin and mouse IgG respectively in samples,
    using the Attana A100® C-Fast system

  • Crude Samples Analysis – Screening and Characterization of Hybridoma Supernatants

    Primarily, to screen mouse IgG antibodies from crude samples, such as hybridoma supernatants,
    for dissociation rates of bound antigen. In addition, to determine detailed kinetic rate constants
    and affinity between the antigen and an antibody selected in the screen

  • Crude Samples Analysis – Direct Off-rate Screening in Supernatants and Lysates

    To determine off-rates of antibodies in mouse hybridoma supernatants and scaffold proteins in
    bacterial lysates using the Attana 200 system and Low Non-specific Binding (LNB) surfaces


  • Capturing of Biotinylated Molecules on the Attana Biotin Sensor Chip Surface

    The Attana Biotin Sensor Chip is designed for versatile and effective capturing of biotinylated molecules (i.e. ligands) on the sensor surface using the homotetrameric protein streptavidin.

  • Immobilization of Antibodies on the Attana Carboxyl Sensor Chip Surface

    This technical note describes the functionality and performance of the Attana Carboxyl Sensor Chip surface for
    immobilization of antibodies.

  • Resistance to non-specific binding of the Attana LNB Carboxyl Sensor Chip Surface

    The Attana LNB Carboxyl Sensor Chip surface is designed for studies of molecular interactions in non-purified samples or samples that have a strong tendency form non-specific interactions. This technical note describes the functionality and performance of the Attana LNB Carboxyl Sensor Chip surface for studies of molecules in crude samples.

  • Characterization of Complex Lectin-Cell Kinetics

    Determine the binding properties of the lectin Concanavalin A (Con A) towards human epidermoid
    carcinoma cells (A431)

  • Glycosylation Profile of Cancer Cells

    Determine the glycosylation profile of cancer cells using a cell based QCM Biosensor assay

  • Early Detection of Clinical Failure

    Evaluate newly developed therapeutic antibodies in a biologically relevant context (cells) in order
    to detect failing candidates at an earlier stage

  • Attana A100 system

    The Attana A100 is a label-free, temperature controlled, continuous-flow system for manual (A100) or automated (A100 C-Fast) analysis of molecular interactions. The system is delivered operation ready and includes: biosensor,
    computer, software and accessories such as syringes, vials and buffer bottles.

  • Attana A200 Product Specification

    The Attana 200 is a dual channel, label-free, temperature controlled, continuous-flow system for manual (Attana 200) or automated (Attana A200) analysis of molecular interactions. The system is delivered operation ready and includes: biosensor, computer, software and accessories such as syringes, vials and buffer bottles.

  • Attana Cell 200 Product Specification

    The Attana Cell 200 is a dual channel, label-free, temperature controlled, continuous flow system for automated
    (Attana Cell A200) or manual (Attana Cell 200) analysis of molecular interactions with cells. The system is delivered
    operation ready and includes: instrument, sensor chips, computer, software and accessories such as syringes, vials
    and buffer bottles.

  • Attana sensor chips and reagents

    The core of the Attana technology lies in the quartz crystal mounted in the sensor chip to allow real time, label-free measurements of molecular interactions. By immobilizing a target molecule to the sensor surface, and flowing an interacting molecule over the surface, the interaction can be studied in real time. The real-time information
    can provide kinetic, affinity and specificity data on the interaction.

  • Feb 5, 2013 Attana Company BrochureCopyright © 2013 Attana AB

    Drug development is financially risky and time consuming. To save money and time, the industry needs to identify and eliminate unsuitable candidates early in the research process. The Attana Cell 200 biosensor system improves the selection process by rapidly identifying promising drug candidates. This enables early and well informed decisions in selecting relevant candidates while reducing unnecessary development cost and saving time.